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FocusWestern Blot

AH diagnostics: Your Science – Our Focus

Dedicated to helping life science and diagnostics laboratories perform their best by providing innovative, high-quality products and expert knowledge.

We offer the most cutting edge technologies within ELISA, flow cytometry, imaging, pathology, protein analysis, qPCR, single-cell analysis and bio appliance.

Representing more than 35 suppliers, our application special-ists are ready to help you with your next project, from assay setup, training and implementation to analysis.

We have 30 years of experience serving the Nordic Region and offices in Copenhagen, Aarhus, Oslo, Stockholm and Helsinki.

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1 SamplePreparation�� 51.1 Cell Lysis ..................................................................................................................................................................................................... 51.2 Protease and Phosphatase Inhibition .................................................................................................................................................. 61.3 Purification ................................................................................................................................................................................................. 71.3.1 Protein, RNA and DNA Purification...................................................................................................................................................... 71.3.2 His-tag and GST-tag Purification ............................................................................................................................................................ 81.3.3 Clontech Purification Products ............................................................................................................................................................101.4 Concentration Measuring .....................................................................................................................................................................12

2 ProteinSeparation�� 132.1 Protein Concentration and Buffer Exchange .....................................................................................................................................132.2 Sample Loading Buffers ........................................................................................................................................................................142.2.1 Reducing and Non-reducing Sample Loading Buffers ....................................................................................................................142.2.2 Real-time, In-gel Protein Stain ...............................................................................................................................................................142.3 Gel Electrophoresis Buffers ..................................................................................................................................................................152.4 Molecular Weight Standards ................................................................................................................................................................152.5 Gel Cast and Run ....................................................................................................................................................................................162.6 Protein Stain .............................................................................................................................................................................................16

3 Blotting�� 173.1 Membranes ..............................................................................................................................................................................................173.2 Blotting Papers ........................................................................................................................................................................................183.3 Transfer Buffer .........................................................................................................................................................................................183.4 Protein Stain for Membranes................................................................................................................................................................19

4 IncubationwithAntibodies�� 204.1 Blocking Solution ....................................................................................................................................................................................204.2 Washing Buffer for Western Blots .......................................................................................................................................................204.3 Direct or Indirect Methods? .................................................................................................................................................................204.4 Primary Antibodies .................................................................................................................................................................................214.5 Secondary Antibodies ...........................................................................................................................................................................214.6 Streptavidin Conjugates ........................................................................................................................................................................23

5 Detection�� 245.1 HRP Substrates ........................................................................................................................................................................................245.2 How to Choose the Right Chemistry for Your Western Blot ........................................................................................................255.2.1 Chemiluminescence ..............................................................................................................................................................................255.2.2 Fluorescence ...........................................................................................................................................................................................25

6 Troubleshooting�� 276.1 Unusual/Unexpected Bands ................................................................................................................................................................276.2 No Bands, Faint Bands or Weak Signal ..............................................................................................................................................286.3 Electrophoretic Problems ......................................................................................................................................................................286.4 High Background ....................................................................................................................................................................................29

Western Blot Overview Western blotting is as a simple and straightforward technique for protein characterization. However, a great number of steps are required to secure optimal results, and we know it is crucial to be confident with the entire process. We have therefore published this magazine to support you in making the right Western blotting choice. Use the above workflow throughout the magazine as a guideline in your daily Western blot work. Simply identify your step of interest and become inspired by our solutions.

Despite Western blotting being a widely used and simple technique, many challenges can still arise. If you come across a prob-lem, turn to the troubleshooting section at the end of the magazine, or contact one of our trained specialists for further support.

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Sample Preparation Protein Separation Blotting AB Incubation Detection ► ► ► ►

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1 Sample PreparationThe first step in obtaining a successful Western blot is to properly prepare the starting material. Prior to sample preparation, it is of the utmost importance to acquire knowledge about the protein location, protein source, hydrophobicity of protein target, etc. Whether you are working with cultured cell lines or tissue samples, sample preparation is often a process of trial and error in which you need to choose the right lysis buffers, enzyme inhibitors and appropriate volumes. Some samples may even require mechanical disruption.

1.1 Cell Lysis

Cell lysis is often performed using a buffer containing detergents and/or denaturants. The choice of buffer depends on cell loca-tion.

From Santa Cruz, we offer:

Protein location Buffer

Whole cell NP-40, RIPA, Triton X-100

Cytoplasmic (soluble) Tris-HCl

Cytoplasmic (cytoskeletal bound) Tris-Triton

Nuclear, mitochondrial, membrane RIPA, Triton X-100, NP-40

Clontech offers the mild, non-denaturing xTractor Lysis Buffer for fast and easy extraction of proteins from bacteria, yeast, mam-malia, and baculo-infected cells. The product is compatible with IMAC resins for quick purification of His-tagged proteins. It takes only 10 minutes!

Cat. no. Description Size

635625 xTractor Buffer 500 mL



635623 xTractor Buffer Kit 10 extractions

NB Clontech products are not available in Denmark and Sweden

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1.2 Protease and Phosphatase Inhibition

Cell lysis is associated with the release of proteases and phosphatases, which in turn negatively affects the Western blot.

UltraCruz® Protease Inhibitor Cocktail Tablet prevents proteolysis from bacterial, yeast, mammalian and plant extracts! Available with or without EDTA. Simply dissolve the tablet in buffer or lysate.


Sc-29130 UltraCruz® Protease Inhibitor Cocktail Tablet 20 tablets

Sc-29131 UltraCruz® Protease Inhibitor Cocktail Tablet, EDTA-free 20 tablets

ProteoGuard™ EDTA-Free Protease Inhibitor from Clontech is a cocktail in solution. Provided in single-use tubes for ease of use.


635672 ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail 100 uL

635673 ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail 10 x 100 uL


For phosphatase inhibitors, use the following mixes from Santa Cruz:

Cat. no. Size Inhibitor mix Target

Sc-45044 1 mL Phosphatase Inhibitor Cocktail A Serine/threonine protein phos-phatases, L-isozymes of alkaline

Sc-45045 1 vial* Phosphatase Inhibitor Cocktail B Tyrosine phosphatases, acid and alkaline phosphatases

Sc-45065 1 mL Phosphatase Inhibitor Cocktail C Alkaline phosphatases, serine/threonine phosphatases

*reconstitute in 1 mL water

Looking for inhibitors that target

specific groups of prote-ases and phosphatases?

Contact us for customized solutions from Santa

Cruz or Enzo.

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1.3 Purification

It can be advantageous to purify proteins by other means than lysing the cells with detergents and denaturants combined with mechanical stress and inhibitors. A large number of methods are available both for purifications prior to Western blotting studies, but also for larger scale downstream purifications.

1.3.1 Protein, RNA and DNA Purification

We offer high-quality purifications for simultaneous experiments on protein, RNA and DNA levels through our long-lasting cooperation with the German company MACHEREY-NAGEL.

• All kits include NucleoSpin® Filters for optimal lysis and rDNase to ensure high-quality RNA.

• High protein yield independent of size, location, modifi-cations, etc.

• The protein fraction is ready-to-use for downstream ap-plications such as SDS-PAGE and Western blot.

• It takes only 30-45 minutes!

NucleoSpin® TriPrep for one-column purification of RNA, DNA and protein from the same sample.

NucleoSpin® RNA/Protein for one-column purification of RNA and protein from a single sample.

NucleoSpin® miRNA for protein isolation, total RNA or, small and large RNA in separate fractions.


740966.50M NucleoSpin® TriPrep 50 preps

740966.250M NucleoSpin® TriPrep 250 preps

740933.50M NucleoSpin® RNA/Protein 50 preps

740933.250M NucleoSpin® RNA/Protein 250 preps

740971.50M NucleoSpin® miRNA 50 preps

740971.250M NucleoSpin® miRNA 250 preps

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1.3.2 His-tag and GST-tag Purification

Protino® is the product line for His-tag and GST-tag purification from MACHEREY-NAGEL. Identify the right IMAC product for your His-tagged proteins. Our products have the highest performance equivalent to market leaders.

Protino® Ni-NTA agarose

Very

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Chelating ligand density Binding capacity Available formats Comments

Low 50 mg/mL Bulk resin, FPLC columns, 96-well plate Change supplier with no need of protocol adaptation


745400.25M Protino® Ni-NTA Agarose 25 mL



745410.5M Protino® Ni-NTA Columns 1 mL 5 preps

745415.1M Protino® Ni-NTA Columns 5 mL 1 prep

745415.5M Protino® Ni-NTA Columns 5 mL 5 preps

745425.1M Protino® 96 Ni-NTA 1 plate

745425.4M Protino® 96 Ni-NTA 4 plates

Protino® Ni-IDA silica


Low 20 mg/mL Dry bulk resin, gravity flow columns, 96-well plate

Can be stored at roomtemperature

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745210.5M Protino® Ni-IDA Resin 5 g




745150.10M Protino® Ni-IDA 150 Packed Columns 10 preps






745300.1M Protino® 96 Ni-IDA 1 plate

745300.4M Protino® 96 Ni-IDA 4 plates

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Protino® Ni-TED silica

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High 10 mg/mL Dry bulk resin, gravity flow columns Can be stored at room tem-perature.


745200.5M Protino® Ni-TED Resin 5 g



745200.600 M Protino® Ni-TED Resin 600 g

745100.10M Protino® Ni-TED 150 Packed Columns 10 preps






Protino® Glutathione Agarose 4B

• Product of market-leading quality – confidently replace your current GST-tag purification with Protino• Available in two formats: ready-to-use FPLC™ columns, and resin for gravity flow or batch purification• Flexible products, which are adjustable in terms of purification method and scale


745500.10M Protino® Glutathione Agarose 4B 10 mL

745500.100M Protino® Glutathione Agarose 4B 100 mL

745510.5M Protino GST/4B Columns 1 mL 5 preps

745515.1M Protino GST/4B Columns 5 mL 1 prep

745515.5M Protino GST/4B Columns 5 mL 5 preps

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1.3.3 Clontech Purification Products

Choose your protein purification products from Clontech. Whether you are working with tagged or non-tagged targets, Clontech offers products for a large number of applications. The categories listed below contain a full range of protein purification prod-ucts.

His-tag purification products• Capturem™ His-tagged Purification Kit• His60 Ni• TALON®

• Magnetic Beads

FLAG-tag purification products• Magnetic DYKDDDDK Immunoprecipitation Kit• Anti-DYKDDDDK Beads

GST-tag purification products• Glutathione-Superflow Resin

Myc-tag purification products• c-Myc Monoclonal Antibody-Agarose Beads• Magnetic Myc Immunoprecipitation Kit

Phosphoprotein enrichment products• Phosphoprotein Enrichment Kit• TALON® PMAC Magnetic Phospho Enrichment Kit• Phosphopeptide Enrichment Spin Columns

Glycoprotein enrichment products• Glycoprotein Enrichment Resin

Antibody purification products• Thiophilic-Superflow Resin

Please contact us for ordering information and further details on the specific products.


NEW!

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Many conventional methods for protein purification require a great deal of time and effort. Immobilized-metal affinity chroma-tography resin columns provide high capacity, but speed and ease of use are compromised due to long equilibration and binding times, as well as slow diffusion of large molecules through the resin bed.

A next-generation membrane technology utilizing spin columns, which overcomes these bottlenecks, is now available from Clon-tech.

Purify His-tagged proteins in 5-15 minutes with the Capturem® His-tagged purification kit

• 4-step protocol at room temperature• Purification of mammalian or bacterial His-tagged proteins• Compatible with both native and denaturing conditions containing DTT, β-mercaptoethanol, TCEP, EDTA or glycerol• High yield and purity

Miniprep Maxiprep High-throughput 96 minipreps

Cat. no. 635710 635713 635714

Yield ~ 100 µg/prep ~ 2.5 mg/prep ~ 100 µg/prep

Sample volume from overnight culture 2-5 mL 10-150 mL 2-5 mL

Preparation time 5 min 15 min

Concentration of eluted protein ~ 0.3-1 mg/mL Up to 4.5 mg/mL ~ 0.3-1 mg/mL

Number of purifications 20 reactions 6 preps 96 preps


NEW!

Equilibration: Add xTractor buffer, spin, discard flow-through

Binding: Load cleared lysate, spin + save flow-through, transfer column to new tube

Washing: Add wash buffer. If needed, add small amount of elution buffer to change imidazole conc. and optimize, spin, save flow-through, transfer column to new tube

Elution: Add elution buffer, spin – elution contains purified and tagged protein

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1.4 Concentration Measuring

After extracting the proteins from the samples and prior to SDS-PAGE, it is important to determine the total protein concentra-tion in each sample. By measuring the concentration, it is possible to ensure the correct amount of protein to be loaded on the gel, thereby minimizing high background and nonspecific antibody binding. Concentration measurements are also useful for semi-quantitative Western blotting – simply load the same amount of protein in each lane and compare the relative levels of the target protein.

DeNovix is a new and innovative instrument for measuring concentrations in an easy and intuitive way. Rapidly measure protein samples before proceeding to Western blot analysis. A 0.5 – 1.0 µL sample is all that is required

DeNovix advantages for protein studies:

• Fluorometer and spectrophotometer in one instrument!• Protein (A280 and labeled) and peptide (A205/215) analysis• High absorbance capabilities: up 1125 mg/mL BSA• Compatible with Qubit and Quant-it fluorescence assays• Built-in cuvette heater controls temperatures for kinetic studies• Small footprint: 20 cm x 33 cm• Weight: 2 kg

Also ideal for quantifying DNA and RNA samples!

Fluorometer: 0.5 mL thin-walled PCR tube

Cuvette: Standard quartz and disposable cuvettes, full spectrum UV-Vis

Microvolume: 0.5 – 1.0 µL, full spectrum UV-Vis

Cat. no.

DS-11 FX + √ √ √

DS-11 FX √ √

DS-11 + √ √

DS-11 √

QFX FLUOROMETER √

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2 Protein SeparationAfter sample preparation, polyacrylamide gel electrophoresis (PAGE) is carried out. Most often, the samples are treated with the negatively charged SDS to unfold and separate the protein content by size. It is also possible to perform non-reducing gel elec-trophoresis to maintain the native state of the protein. Furthermore, it is helpful to consider the polyacrylamide gel percentage, which is dependent on protein size – larger proteins needs lower acrylamide concentration and vice versa.

2.1 Protein Concentration and Buffer Exchange

Prior to protein separation, it is often necessary to concentrate proteins and remove contents, which interferes with gel electro-phoresis.

The Afyon SDS-PAGE Sample Preparation Kit removes unwanted buffer contents and concentrates protein samples to save you hours in the lab.

Superior advantages:

• Contaminants (GuHCl, urea, ammonium, sulfate etc.) are efficiently removed• It takes less than 10 minutes• Normalization of protein concentration – final filtrate contains exactly 5 µg• Non-toxic• Compatible with SDS-PAGE as well as chemiluminescent and fluorescent Western blotting

Extract sample

Add 20 µL Afyon Resin to sample*, vortex, centrifuge, remove supernatant (< 5 min)

Suspend pellet in loa-ding buffer, centrifuge with spin filter (< 5 min)

Filtrate contains exactly 5 µg to load directly on gel

Run gel

*Compatible with dilute samples and samples solubilized in GuHCI, detergents, etc.

► ►► ►

Afyon protocol in just 10 minutes:


K-02101-010 Afyon SDS-PAGE Sample Preparation Kit 10 reactions

K-02101-025 Afyon SDS-PAGE Sample Preparation Kit 25 reactions

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2.2 Sample Loading Buffers

Most often, the prepared samples can be stored at -20°C until use. Prior to gel electrophoresis, the samples are prepared with either a reducing or non-reducing loading buffer, which ensures the right viscosity, color for monitoring the loading and running, denaturation, reduction and pH maintenance.

2.2.1 Reducing and Non-reducing Sample Loading Buffers

Avoid wasting time and use pre-mixed loading buffers for SDS-PAGE. Simply add your sample to the loading buffer, boil and load. Samples can remain at room temperature for immediate use, or they can be stored at 4°C or -20°C for later analysis.

Choose between reducing and non-reducing loading buffers – both are compatible with staining and Western blotting applica-tions.

Cat. no. Description Volume

R-03018-B10 Non-reducing protein sample loading buffer (2x) 1 mL

R-03018-B50 Non-reducing protein sample loading buffer (2x) 5 mL

R-03019-B10 Reducing protein sample loading buffer (2x) 1 mL

R-03019-B50 Reducing protein sample loading buffer (2x) 5 mL

2.2.2 Real-time, In-gel Protein Stain

After gel electrophoresis, it is important to visualize your protein bands to ensure that the separation is successful. This is known to be a time-consuming and inconvenient process.

Visio in-gel protein stain from Advansta helps you to quickly and easily detect protein bands while running your gel.

• Instant results: follow the visible bands while running the gel• Sensitive: detect down to 10 ng per band• Fast: heat your samples with Visio for 10 minutes and go• Compatible: use with both reducing and non-reducing gels. Analyze your

results with downstream applications*


K-11053-300 Visio Real-Time Protein Stain, 100 lanes or 10 gels 0.3 mL

K-11053-B30 Visio Real-Time Protein Stain, 1,000 lanes or 100 gels 3 mL

*Compatible with mass spectrometry. The product has not been validated with downstream Western blotting, but many customers have had success with it.

1) Mix Visio with protein sample

2) Incubation at 98 °C for 10 min, vortex and centrifuge briefly – add reducing buffer for reducing gels

3) Load

4) Run

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2.3 Gel Electrophoresis Buffers

To ensure the right pH, electrical conductivity and a negative charge on the proteins, the right composition of running buffer should be used.

Save time with Avant Buffer Pouches. By adding just 500 mL of deionized water and mixing, you will have a freshly made, ultrapure buffer for your SDS-PAGE, which ensures reproducible results.


R-01038-020 PBS (concentration 1x at 500 mL) 20 pouches

R-01039-020 TBS (concentration 1x at 500 mL) 20 pouches

R-01037-020 Tris-Glycine PAGE running buffer (concentration 1x at 500 mL) 20 pouches

R-01036-020 Tris-Glycine SDS-PAGE running buffer (concentration 1x at 500 mL) 20 pouches

2.4 Molecular Weight Standards

In addition to the Western blot signal, knowledge of the protein size is very useful, which can be confirmed with molecular weight standards.

From Santa Cruz, we offer a number of standards, which are provided in SDS-PAGE loading buffer for direct use on your gel:

• Available in low, broad and high range. • Use Cruz Marker™ Molecular Weight Standards for visualization on the final Western blot after incubation with the Cruz

Marker™-compatible secondary antibodies for Western blotting. Choose between either HRP or AP conjugated secondary antibodies.


Sc-2035 Cruz Marker™ Molecular Weight Standards sufficient for 50 gels

Sc-2361 Broad Range Markers 500 µL

Sc-2362 High Range Markers 500 µL

Sc-2360 Low Range Markers 500 µL

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2.5 Gel Cast and Run

Electrophoresis performance is dependent on a uniformly cast gel and a high-quality running apparatus.

UltraCruz® Electrophoresis Cell – simple, safe and easy

• Two-in-one cell suitable for both gel casting and run. • Can run up to 10 or 15 samples. • Power turns off automatically when lid is opened• High resolution separation

Cat. no Description

Sc-201625 UltraCruz® Electrophoresis Cell

2.6 Protein Stain

Check the quality of protein resolution on your gel or the protein transfer on your membrane by staining.

Visualize total protein on your gel or blot with the non-toxic fluorescent AdvanStain Scarlet to detect down to 1 ng on laser or CCD imaging systems. Destain your gel and you are ready for downstream Western blotting. Excitation wavelengths include blue, green, violet and UV, while maximum emission wavelength is 610 nm.

1D and 2D gels stained with excellent sensitivity, large dynamic range, low background and no speckling!


K-11072-B50 AdvanStain Scarlet Kit, sufficient for 20 minigels 5 mL

K-11072-C25 AdvanStain Scarlet Kit, sufficient for 100 minigels 25 mL

Fix (1 hour)

Stain (1 hour)

Wash (30 min)

Acidify (30 min) Image

Fast protocol:

► ►► ►

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3 BlottingBlotting is carried out to transfer proteins from the gel to a solid membrane. The blotting is performed by assembling filter papers, a membrane and a gel into a sandwich. An electrical field allows the proteins from the gel to wander onto the membrane.

3.1 Membranes

Choose between nitrocellulose and PVDF membranes. We recommend pre-cut membranes for ease of use. Alternatively, cut your membranes from a roll for an economical solution.

Use WesternBright transfer membranes for high performance and low background levels. WesternBright membrane characteris-tics:

WesternBright PVDF-FL WesternBright PVDF-CL WesternBright NC

Pore size 0.22 µm 0.22 µm 0.22 µm, 0.45 µm

Thickness 135 µm 140 µm 150 µm

Binding interaction Hydrophobic Hydrophobic Hydrophobic, electrostatic

Typical protein binding capacity

300 µg/cm2 300 µg/cm2 200 µg/cm2

Detection methods

FluorescenceTotal protein stain

Total protein stainChromogenicChemiluminescence

ChemiluminescenceDirect stainingRadiolabelingColorimetric

Immobilization methods

Electrophoretic transfer of proteins Electrophoretic transfer of proteins Electrophoretic transfer of proteinsProtein dot and slot blotting

Recommended applications

Western transfer with fluorescent detection

Western transfer with chemiluminescent detection

Western transfer with chemiluminescent detectionColony/plaque lifts

Available asPre-cut membranesMembrane rolls

Pre-cut membranesMembrane rolls

Pre-cut membranesMembrane rolls


L-08003-010 Pre-cut WesternBright NC 0.22 um, 8 x 10 cm 10 sheets

L-08002-010 Pre-cut WesternBright NC 0.45 um, 8 x 10 cm 10 sheets

L-08004-010 Pre-cut WesternBright PVDF-CL, 7 x 9 cm 10 sheets

L-08001-010 Pre-cut WesternBright PVDF-FL, 7 x 9 cm 10 sheets





L-08006-001 WesternBright NC membrane roll, 0.22 µm, 30 cm x 3 m 1 roll

L-08007-001 WesternBright PVDF-FL membrane roll, 0.22 µm, 26 cm x 3.3 m 1 roll

L-08008-001 WesternBright PVDF-CL membrane roll, 0.22 µm, 26 cm x 3.3 m 1 roll

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3.2 Blotting Papers

Standardized, pre-cut blotting papers save you time during sandwich assembly.

We offer blotting papers with useful advantages:

• Pre-cut papers available in two sizes• Uniform thickness ensures reproducible transfers without introducing artifacts or background noise


L-07045-060 Blotting papers, 7 x 9 cm 60 sheets

L-07056-060 Blotting papers, 8 x 10 cm 60 sheets

3.3 Transfer Buffer

It is important to choose a high-quality transfer buffer to optimize the detection of low-abundance proteins, the transfer of high-molecular weight proteins and to save time.

Optimize the transfer from gel to membrane with Advansta’s proprietary FLASHBlot Transfer Buffer:

• Ideal for low-abundance protein samples and post-translationally modified proteins• Transfer proteins of all sizes in less than 20 minutes

CEA (high MW) and GAPDH (low MW) Western blots. The proteins were transferred for 15 minutes using a FLASHBlot buffer and 60 minutes with a Towbin buffer, respectively. Transfers for both CEA and GAPDH were most efficient when using FLASHBlot.


R-03090-D25 FLASHBlot Transfer Buffer, 50x 250 mL

R-03090-D50 FLASHBlot Transfer Buffer, 50x 500 mL

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3.4 Protein Stain for Membranes

Before proceeding with antibody incubation, it is advantageous to check the quality of the protein transfer.

Advantages with AdvanStain Ponceau:

• Stains your membrane in less than 5 min.• Destain the membrane just as easily• Following destaining, the membrane is ready for incubation with antibodies


R-03021-D50 AdvanStain Ponceau 500 mL

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4 Incubation with AntibodiesOnce the proteins have been transferred to a membrane, it needs to be blocked, incubated with a primary antibody, washed, in-cubated with a secondary antibody and then washed a final time. Instead of using both primary and secondary antibodies, direct detection can, in many cases, be more suitable.

4.1 Blocking Solution

Avoid non-specific binding of antibodies by using an appropriate blocking solution.

AdvanBlock-PF offers:

• Non-protein blocking solution• No cross-reactivity with primary antibodies as seen with nonfat dry milk, BSA or casein• Compatible with both fluorescent and chemiluminescent detection• Optimized for use with WesternBright fluorescent secondary antibodies


R-03023-D20 AdvanBlock-PF Blocking Solution, 5x 200 mL

4.2 Washing Buffer for Western Blots

After each incubation step with antibodies, the blot needs to be washed with an appropriate washing buffer.

Use AdvanWash washing solution whether you are performing fluorescent or chemiluminescent Western blotting. The solutions can be used with any Western blotting systems, but are optimized for use with WesternBright chemiluminescent HRP substrates, WesternBright MCF fluorescent Western blotting kit and WesternBright MCF-IR kits.

• Reliable: Quality-controlled for reproducible results• Versatile: Compatible with chemiluminescent and fluorescent Western blots• Convenient: Save time using pre-made buffers


R-03024-D50 AdvanWash Washing Solution, 10x 500 mL

R-03100-D50 AdvanWash-IR Washing Solution, 10x 500 mL

4.3 Direct or Indirect Methods?

Direct detection relies on a single primary antibody containing a detectable label, while indirect detection requires both a primary antibody as well as a secondary antibody. In indirect targeting, the secondary antibody contains the label.

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Indirect Direct

Advantages

• Signal amplification – multiple secondary antibodies bind to primary antibody

• Versatile – same secondary antibody can be used on different primary antibody of same species

• Fewer steps• Less potential for non-specific binding

Disadvantages• Risk of non-specific binding• More steps

• Can be more costly• Labeling of antibody can affect the immunoreactivity

4.4 Primary Antibodies

When performing Western blots, it is important to choose antibodies that recognize only the epitopes of interest.Follow the table below for advice on whether to choose monoclonal or polyclonal antibodies:

Monoclonal antibodies Polyclonal antibodies

Recognition of epitope • Recognize a single epitope • Recognize multiple epitopes within same antigen

Sensitivity • Less sensitive • More sensitive – good for low abundance antigens

Cross-reactivity• Less likely• Potential for recognizing other antigens

with the epitope

• More likely • Higher background than monoclonal antibodies• Can cross-react with multiple animal species

Variability • Very stable batches from same hybridoma • Variability-prone between batches

Tolerance* • Less tolerant • More tolerant

*Tolerance against differing conditions, such as denaturing conditions, modifications or presence of polymorphism

4.5 Secondary Antibodies

For indirect detection, the blot is treated with secondary antibodies after primary antibody incubation. The primary antibody species determines the choice of secondary antibodies. For chemiluminescence, secondary antibodies are labeled with HRP, while for fluorescence they are labeled with a fluorophore. We offer secondary antibodies from a variety of species with different conjugates.

HRP-conjugated secondary antibodies for chemiluminescent detection:


R-05071-500 Goat anti-mouse HRP-conjugated secondary antibody 500 µL

R-05072-500 Goat anti-rabbit HRP-conjugated secondary antibody 500 µL

R-05073-500 Goat anti-human HRP-conjugated secondary antibody 500 µL

R-05074-500 Goat anti-chicken HRP-conjugated secondary antibody 500 µL

R-05075-500 Goat anti-rat HRP-conjugated secondary antibody 500 µL

R-05076-500 Goat anti-guinea pig HRP-conjugated secondary antibody 500 µL

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Fluorescent secondary antibodies, excitable with visible and near infrared light:

Cat. no Description, visible light Size

R-05051-050 Goat anti-rabbit IgG APC 50 µL

R-05051-250 Goat anti-rabbit IgG APC 250 µL

R-05052-050 Goat anti-mouse IgG RPE 50 µL

R-05052-250 Goat anti-mouse IgG RPE 250 µL

Cat. no. Description, infrared light Size

R-05054-250 Goat anti-rabbit IgG IR700 250 µL

R-05055-250 Goat anti-mouse IgG IR700 250 µL

R-05056-250 Goat anti-human IgG IR700 250 µL

R-05057-250 Goat anti-chicken IgY IR700 250 µL

R-05058-250 Goat anti-rat IgG IR700 250 µL

R-05059-250 Goat anti-guinea pig IgG IR700 250 µL

R-05060-250 Goat anti-rabbit IgG IR800 250 µL

R-05061-250 Goat anti-mouse IgG IR800 250 µL

R-05062-250 Goat anti-human IgG IR800 250 µL

R-05063-250 Goat anti-chicken IgY IR800 250 µL

R-05064-250 Goat anti-rat IgG IR800 250 µL

R-05065-250 Goat anti-guinea pig IgG IR800 250 µL

Secondary antibodies for a large selection of species:

From Santa Cruz Biotechnology we offer a wide variety of conventional secondary antibodies for a large selection of species, raised in either rabbits, goats, donkeys, bovines, mice or chickens.

• Anti-Goat• Anti-Mouse• Anti-Rabbit

• Anti-Rat • Anti-Bovine • Anti-Cat

• Anti-Chicken• Anti-Dog • Anti-Guinea Pig

• Anti-Armenian Hamster • Anti-Horse• Anti-Human

• Anti-Monkey • Anti-Swine• Anti-Turkey

• Anti-Sheep • Anti-Syrian Hamster• Anti-Donkey

Available conjugated to either an enzyme, biotin or fluorophore.

Cruz Marker™-compatible Western blotting secondary antibodies and Cruz Marker™ Molecular Weight Standards.

Cruz Markers™ are provided for use as internal molecular weight standards for chemiluminescence Western blotting applications, and they are ideal for approximating the molecular weight of target proteins while allowing researchers to visualize the final mo-lecular weight ladder of six bands clearly and consistently on the final Western blot film.

Cruz Marker™ compatible secondary antibodies recognize an epitope common to each of the Cruz Marker™ molecular weights. Pre-adsorbed secondary antibodies are recommended for Western blotting of immunoglobulin-rich tissues and cells.

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Cruz Marker™ Molecular Weight Standards*

Description Cat. no Size

Cruz Marker™ Molecular Weight Standards

sc-2035 200 µL

*Must be used in connection with Cruz Marker™-compati-ble Western blotting secondary antibodies.

4.6 Streptavidin Conjugates

The strong interaction between streptavidin and biotin can be utilized for Western blot-ting. Using streptavidin conjugates enables fluorescent detection of biotinylated proteins.

Fluorescent streptavidin conjugates:


R-05011-050 AdvanFluor APC-Streptavidin conjugate 50 µL

R-05012-050 AdvanFluor BPE-Streptavidin conjugate 50 µL

R-05011-200 AdvanFluor APC-Streptavidin conjugate 200 µL

R-05012-200 AdvanFluor BPE-Streptavidin conjugate 200 µL

We offer over 70,000 monoclonal and polyclonal primary and secondary Western blot antibodies from our suppliers. Antibodies for cancer research, cardiovascular research, drug discovery, immunology, metabolism, neuroscience research, etc.

Cruz Marker™-compatible Western blotting secondary antibodies*Description HRP conjugate AP conjugate HRP conjugate

Pre-adsorbedAP conjugatePre-adsorbed

Bovine anti-goat IgG sc-2378 sc-2381 sc-2384 sc-2387

Donkey anti-goat IgG sc-2033 sc-2037 sc-2304 sc-2310

Bovine anti-mouse IgG sc-2380 sc-2383 sc-2386 sc-2389

Donkey anti-mouse IgG sc-2318 sc-2320 sc-2306 sc-2312

Goat anti-mouse IgG sc-2031 sc-2047 sc-2302 sc-2308

Bovine anti-rabbit IgG sc-2379 sc-2382 sc-2385 sc-2388

Donkey anti-rabbit IgG sc-2317 sc-2319 sc-2305 sc-2311

Goat anti-rabbit IgG sc-2030 sc-2034 sc-2301 sc-2307

Goat anti-rat IgG sc-2032 sc-2036 sc-2303 sc-2309

Rabbit anti-mouse IgG sc-358917 sc-358923

*Are supplied at 200 µg in 0.5 mL, to be used in Western blotting at a dilution of 1:500–1:5000.

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5 DetectionThe detection method is dependent on the label of the antibodies. Using a fluorescence dye enables detectable excitation by light. For chemiluminescent detection, HRP-labeled antibodies are treated with substrate, which produces the detectable signal.

5.1 HRP Substrates

Most Western blot users have special needs for HRP substrates, including high sensitivity, long-lasting signal, fast detection and higher dynamic range.

Choose the right product for the chemiluminescent detection of your target proteins.

Substrate Use Sensitivity Signal duration

WesternBright ECL Sensitive, economical choice for routine Western blotting Low picograms 6-8 hours

WesternBright Quantum Extended dynamic range for detecting low and high abun-dance proteins within the same blots

Mid femtograms 10-24 hours

WesternBright Sirius Low abundance protein detection Low femtograms 6 hours


K-12045-D20 WesternBright ECL 200 mL

K-12045-D50 WesternBright ECL 500 mL

K-12049-D50 WesternBright ECL spray 500 mL

K-12042-D10 WesternBright Quantum 100 mL

K-12042-D20 WesternBright Quantum 200 mL

K-12043-D10 WesternBright Sirius 100 mL

K-12043-D20 WesternBright Sirius 200 mL

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5.2 How to Choose the Right Chemistry for Your Western Blot

The best chemistry for your Western blot depends on the type of information you are looking for.

5.2.1 Chemiluminescence

Chemiluminescent Westerns are relatively easy to do and can be extremely sensitive; substrates are available to detect proteins in the femtogram range.

“Chemi” is a convenient chemistry if proteins of interest differ significantly in molecular weight and are easily resolved by gel electrophoresis.

Advantages: Disadvantages:

• Sensitive• Easy, familiar chemistry• Compatible with film or digital imaging

• Semi-Quantitative• Signal dependent on enzyme kinetics• Single protein only, loading controls require stripping and

re-probing Use Chemiluminescence to:

• Detect a single protein• Assay for presence/absence of a protein• Measure antibody responses• Follow protein purification• Detect low abundance proteins

5.2.2 Fluorescence

Fluorescent Western blotting uses secondary antibodies conjugated to fluorescent dyes. The amount of light emitted from excited fluorophores is proportional to the amount of target protein on the membrane. This allows for true quantitative analysis.

Using fluorophores with different emission spectra, one can detect multiple proteins on a blot simultaneously (multiplexing). Loading controls can be measured on the same blot as your protein of interest, along with post-translational modifications without stripping and re-probing.

Fluorescent Westerns use either Infrared or Visible fluorophores.

Advantages: Disadvantages:

• Multiplex capability• Increased quantitative accuracy• Fluorescent label stability allows blots to be stored and

re-imaged later

• Can be less sensitive than chemiluminescence• Membrane auto-fluorescence can increase background

Advantages of Visible Fluorescent Detection

If you need to study three proteins at once, visible fluorescence is the obvious choice. Visible fluorescence also offers flexibility. There are many choices of fluorophores available for visible detection. If you are already using (spectrally separated) visible fluoro-phores for other experiments, adapting these for use in fluorescent Western blots is easy.

Chemiluminescence. One signal. One protein

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Use Fluorescence to:

• Detect multiple proteins simultaneously• Study post-translational modifications• Have same-blot loading control• Have in-lane normalization• Perform quantitative Westerns

The Azure c Series Imaging Systems The Azure Biosystems cSeries offers 6 unique gel imaging systems. Select the one that fits your applications now, and upgrade later when your needs change.

• A total solution for Western Blot Imaging and Gel Documentation: The cSeries is a multichannel, multimodal imager, with IR, visible light, and UV excitation channels.

• Compact, integrated design: A unique design and integrated tablet computer gives this system a small foot-print, saving you room for other tools on the bench top.

• Fully upgradable: All models can be easily upgraded at a later time to accommodate additional applications.

• 8.3MP CCD camera for sensitive detection of chemiluminescence: 5 levels of binning and a Chemi Blot Shelf to bring the blot closer to the detec-tor, if needed.

• 16 bit camera: c-series has a wide dynamic range, preventing saturation and keeping results in linear range.

• Deep Peltier cooling of the camera: Absolute and Regulated cooling improves image quality by reducing the noise in the image.

• AzureSpot Analysis Software: Analysis tool for 2D densitometry, automatic lane and band detection, back-ground correction, normalization, molecular weight calculation, and annotation.

c600 √ √ √ √ √ √c500 √ √ √ √ √c400 √ √ √ √ √c300 √ √ √ √c200 √ √ √c150 √ √ √

Multiplex Fluorescence.Multiple Proteins

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6 Troubleshooting

6.1 Unusual/Unexpected Bands

Problem Cause Solution

Lower MW than expected

• Digestion/cleavage of target • Use fresh sample and protease inhibitors

• Splice variant exist or other proteins bear similar epitope

• Consult literature and use appropriate controls• Try another antibody

Slightly higher MW than expected

• Potential for glycosylated protein or small amino acid modifications

• Use enzymes to remove the modification• Check amino acid sequence and literature

Significantly higher MW than expected

• Dimers/multimers/protein-protein interactions oc-cur due to inefficient reduction and denaturation

• Use fresh DTT or β-mercaptoethanol for samples + reheat before repeating experiment

• Prepare new samples with fresh loading buffer

• Changed protein expression during over-passaged cell line

• Use earlier passages, include positive control

Multiple bands

• Excessively high concentration of primary anti-body or occurrence of cross-reactivity with similar epitopes on other proteins

• Use affinity-purified primary antibody• Optimize primary antibody concentration• Try another• Check antibody specificity with blocking peptide

• Non-specific binding of secondary antibody

• Optimize secondary antibody concentration• Use affinity-purified secondary antibody• Check non-specific binding by repeating immunode-

tection with secondary antibody alone

Blurry/diffuse bands

• Voltage too high during gel run • Run gel at lower voltage

• Slow migration • Increase voltage, ensure proper buffer preparation

• Incorrect heating of sample • Ensure heating to 90 °C for 2 min prior to loading

• Old SDS in sample buffer • Use fresh SDS for sample buffer

• Incorrect buffer composition • Use fresh buffer

• Bubbles trapped between gel and membrane during transfer • Carefully remove air bubbles

Smiling bands (not flat)

• Overheated gel due to fast running conditions • Optimize electrophoresis conditions• Run gel at 4 °C

White bands • Too much protein loaded • Decrease amount of sample

• Repeat with dilution series of sample

• Antibody concentration too high • Optimize antibody concentrations

Streaking in lanes

• High salt concentration in sample • Decrease salt concentration in sample buffer

• Sample concentration too high/insufficient SDS • Increase dilution/add more SDS

Lateral sprea-ding of bands

• Diffusion of samples from well to well • Minimize loading time

Band distortion

• Incomplete polymerization of gel around wells • Increase TEMED/AP

• High pressure applied to gel during pouring • Loosen screws on the gel assembly apparatus

• Particulate matter in gel • Filter and mix gel reagents prior to gel preparation

• Excessive/uneven heating of gel • Decrease running voltage/apply cooling during run

• Bubbles in gel due to dirty plates• Wear gloves during plate handling• Clean plates thoroughly with ethanol and deionized

water

• Bubbles in gel from air introduced from pouring device • Do not expel entire volume of gel mix

• Bubbles under comb • Insert comb at an angle and reposition before gel solidification

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6.2 No Bands, Faint Bands or Weak Signal


Sample preparation

• Inefficient extraction method• Use other extraction methods• Include positive control

• Low expression levels of protein• Load more protein, concentrate extracted proteins,

pool multiple samples

• Degraded protein during degradation • Use fresh protease inhibitor

Inadequate transfer

• Wrong transfer buffer composition, too much methanol in buffer

• Check protocol, decrease methanol concentration

• Large protein requires more time and/or current • Run for longer time and/or with higher voltage

• No contact between gel and membrane • Check fiber pad thickness, replace if too thin

• Protein has transferred through the membrane • Use two membranes

Inefficient bin-ding of primary antibody

• Old/weak antibody, low affinity of antibody for protein

• Increase primary antibody concentration, purchase fresh antibody, reduce number of wash steps

• Weak cross-reactivity with species of interest • Try other primary antibody source

• Antibody removed with washing steps• Decrease number of washes, decrease salt concentra-

tion in wash buffer, reduce washing time

• Antigen masked by blocking agent • Use alternative blocker• Try lower concentration of blocker

Inefficient bin-ding of seconda-ry antibody

• Inappropriate secondary antibody • Use antibody directed against primary antibody spe-cies

• Insufficient antibody concentration/antibody too old

• Purchase new antibody• Increase dilution

Inactive conju-gate/substrate

• Old/unstable reagents • Check conjugate and substrate for signal• Purchase new reagent

• HRP inactivation by sodium azide • Avoid sodium azide

Detection methods

• Old detection reagent/improper storage of detec-ting reagent • Purchase new reagent

• Exposure time too short • Test different exposure times

6.3 Electrophoretic Problems


Slow/No gel polymerization

• Insufficient TEMEP/AP • Increase amount of TEMEP/AP

• Old AP solution • Prepare fresh AP and store only for a few days at 4 °C

• Polymerization inhibition due to oxygen • Layer gel with isopropanol prior to pouring stacker

Too fast gel polymerization

• Excessive amount of TEMED/AP • Decrease amount of TEMED/AP

Very long run time

• Concentration of running buffer too high • Check protocol/dilute buffer

• Insufficient current • Increase voltage

Running time unusually short

• Buffer excessively diluted • Check protocol/replace buffer

Smiling dye front

• Migration too fast • Decrease voltage

• Heat generation • Run with cooling

Slanted dye front

• Bubble between glass plates at the bottom of the gel

• Hold gel at an angle, place corner into lower buffer chamber and slowly move to normal position

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6.4 High Background


Membrane blocking is insufficient

• Biotin in milk incompatible with streptavidin system/milk contains antigen of interest

• Try another blocking agent

• Milk solution excessively diluted • Increase milk solution

• Blocking time too short • Increase incubation time

• Some detergents are not as effective at cold temperatures

• Incubate for 1 hour at room temperature instead of overnight at 4 °C

Inappropriate wash conditions

• Insufficient detergent concentration• Use stronger detergents/increase detergent concen-

tration

• Insufficient washing• Increase duration of washing steps, use larger volume

of washing buffer, increase number of washing steps• Try other incubation temperatures

Reagant contamination

• Bacterial/fungal growth in buffers• Check turbidity in buffers• Prepare new buffers

Membrane choice

• PVDF membranes may have higher background than nitrocellulose

• Try nitrocellulose membranes

• High autofluorescence in membrane • Use low autofluorescence PVDF membranes for fluo-rescent Western blots

• Membrane has dried out • Ensure hydration during all steps by using sufficient volumes and agitation

Overexposed image

• Exposure time too long • Reduce exposure time, increase antibody dilutions• Load less sample

Non-specific bin-ding of primary or secondary antibodies

• Antibody concentration too high/antibody not affinity purified

• Decrease antibody concentration/try monoclonal or affinity-purified antibody/optimize incubation times

• Too much protein on gel • Load less protein

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Contact

Denmark

+45 8745 9010 [email protected]

Sweden

+46 08 680 08 [email protected]

Norway

+47 2323 [email protected]

Finland

+358 8 010 325 [email protected]

blå tynd streg hvid alm. streg blå alm. streg pink tynd streg




AH diagnostics as • Gammel Viborgvej 11A • DK-8381 Tilst • Tel.: +45 8745 9010 • [email protected] • www.ahdiagnostics.dkAH diagnostics AB • Vallgatan 9 • SE-170 67 Solna • Tel.: +46 (0)8 680 0845 • [email protected] • www.ahdiagnostics.se

AH diagnostics Oy • Viikinkaari 4 • FI-00790 Helsinki • Tel.: +358 (0)10 325 3000 • [email protected] • www.ahdiagnostics.fiAH diagnostics as • Fjellgata 1 • NO-0566 Oslo • Tel.: +47 2323 3260 • [email protected] • www.ahdiagnostics.no

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