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Western Blot Tips and Tricks

Filling the Gap Between Art and Science

Instructions for Viewers

Webinar Series

July 30, 2014

Brought to you by the Science/AAAS Custom Publishing Office

Sponsored by:

Participating Experts

Pier Giorgio Righetti, Ph.D.

Polytechnic University of Milan

Milan, Italy

Biji T. Kurien, Ph.D.

University of Oklahoma Health

Sciences Center

Oklahoma City, OK

Webinar Series

Nick Thomas, Ph.D.

GE Healthcare

Cardiff, Wales



July 30, 2014

Pier Giorgio Righetti

Department of Chemistry, Materials and

Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, 20131

Milano, Italy

• Most likely is the protein load • Typically as low as 1 ng, the detection limit of silver staining • At such low loads, blots can be poorly reproducible, due to:

Antigen losses already during sample homogenization and centrifugation of cell debris

Losses due to adsorption onto glass and plastic walls of sample containers

Losses due to sample adsorption by the polyacrylamide gel fibers in SDS-PAGE or IEF in IPGs or 2D mapping

Incomplete electrophoretic transfer to the blotting membrane • Due to all above accidents, it is no wonder that for trace antigens

Western blot analysis might be unsuccessful!

• Subtraction approach: immuno-depletion of the 20 high-abundance proteins in

biological fluids (notably serum, urines, cerebrospinal fluid). The problems? • Capability of handling only minute sample volumes (max 100 µL) • Sample dilution after the depletion process • Therefore only very poor gain in visibility of trace proteins

• Enrichment approach: enabling capture of specific classes of proteins, e.g.

glycoproteins, phosphoproteins or an “ecumenic” approach permitting capture of all sample proteins by implementing a substantial reduction of the dynamic range. This approach is the “combinatorial peptide ligand library (CPLL) methodology”. Its advantages:

• Capability of handling very large sample volumes (up to 1 L) • Simultaneous reduction of high-abundance proteins (HAP) and high enrichment of

low-abundance proteins (LAP) • Increment of visibility for LAPs of up to 3 to 4 orders of magnitude!

The hexapeptides are

bound to an organic

polymer,

poly(hydroxymethyl

acrylate) all throughout

the core of the pearls.

Some 64 million diverse

baits are present on the

beads, enough to

capture just about any

protein expressed in

any proteome!

Note that the

hexapeptides terminate

with a D-amino acid!

Initial sample (large dynamic range)

Ligand library

Loading

a b c d

Adsorption

FT fraction

Wash away unbound sample

Recover bound protein

Collected sample (diminished dynamic range)

1

2

3

4

Ctrl.

FT

• When complex samples are incubated with the beads, proteins bind to their specific ligands.

• High abundance proteins quickly saturate their specific ligands.

• High abundance proteins cease binding upon reaching their saturation point.

• Unbound proteins in excess, are removed during wash step.

• Medium and low abundance proteins continue to bind to their ligands.

Reduction of dynamic range by CPLL Beads

• Simultaneous dilution of HAP and concentration of LAP;

• In a single and reproducible step;

• No species depletion is contemplated with this method;

• “Normalization” exclusively achievable using bead libraries of a very large diversity;

• A controlled sample-to-bead ratio allows a given reduction in dynamic range of the proteome.

Range of Detection

Visibility of 2 SAA spots starts at 5-10 ng/μL concentration

Zoomed areas of control samples - untreated

0 ng/μL SAA

160 ng/μL SAA 80 ng/μL SAA 40 ng/μL SAA


20 ng/μL SAA

8-16% Tris-HCl gels

Visibility of 2 SAA spots starts at 1 ng/μL concentration (note that the OD of one of this spots is similar to the density of the 80 ng/μL in the untreated sample of the previous slide)!

Zoomed areas of eluted samples – treated with CPLLs

16% Tris-HCl gel

0 ng/μL SAA



20 ng/μL SAA

Figure 3 - Fasoli et al.

3 pH 103 pH 10

1

2

3

4

5

250

150

50

37

25

20

15

10

75

kDa

100

3 pH 10

1

2

3

4

5

1L

2R

6

7

8

3 pH 10

10

10

Blot

100

75

50

37

2520

15

10

150

250

kDa

Fig. 2 – Fasoli et al.

New allergenic proteins:

• Vicilin

• globulin-2

• endo-chitinase

• thioredoxin

• trypsin inhibitor

Blotted proteins revealed with radio-iodinated antibodies (IgE) against sera of 3 different maize-allergic patients

Zea-mays B73

Lipid transfer protein (LTP)

Control: only one antigen Pers a 1; After CPLL treatment: five more: profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone

reductase-like protein

Known allergens in control, untreated sample: Mus a 1 (profilin), Mus a 2 (class I chitinase), Mus a 4 (thaumatin-like protein), and Mus a 5 (β-1,3-

glucanase).

New allergens after CPLL treatment: a pectinesterase and a superoxide dismutase

Control: 374 unique gene products

CPLL Treated: 2855 unique proteins

Known allergens in control: no one reported so far;

New allergens after CPLL treatment: non-specific lipid transfer protein, superoxide dismutase, germin-like protein and profilin.


Sponsored by:




Milan, Italy



Sciences Center

Oklahoma City, OK

Webinar Series

Nick Thomas, Ph.D.

GE Healthcare

Cardiff, Wales



July 30, 2014

Tips and challenges in

western blotting

Western blotting

Transfer of protein patterns from

gel to microporous membranes

Electrophoretic-Towbin, Burnette

Burnette, to retain “geographic”

naming tradition initiated by Ed

Southern’s paper

Advantages of transfer to

membrane

Wet membranes are pliable and

are easy to handle

This allows easy accessibility of

proteins immobilized on membrane

to different ligands

Only small amount of reagents

required for transfer analysis

Advantages (cont’d)

Multiple replicas of a gel possible

Prolonged storage of transferred

patterns

Same protein transfer for multiple

successive analyses

http://dna-protein.blogspot.com/2012/07/the-good-bad-

and-ugly.html

Considerations for a

good western blot

Proper sample preparation

Choice of right lysis buffer

Proper gel preparation

Right electrophoresis and

transfer conditions

Sample preparation

From cultured cells

Washed with PBS to remove

media

Remove PBS prior to cell lysis

Salt contamination causes

horizontal streaking in 2 DE

Westermeier R. Practical Proteomics, 2007;

Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Eliminating viscosity from

DNA in crude extracts

Treat extracts with protease

free nuclease (Benzonase)

Shearing with sonication

Vigorous vortexing of heated

sample

Protein solubilization:

Choice of Lysis buffers

Laemmli (cell extracts)

Urea/thiourea – for 2D gels

(small HSP)

RIPA (smooth muscle proteins)

Personalize lysis buffer

Peach et al, 2013. Methods in Mol Biol 869, 37-47.

SDS to protein ratio

1.4 µg SDS per 1µg of protein

Recommended SDS to protein

ratio - 3:1

Protein concentration should be

assayed to prevent inadequate

protein to sample buffer ratios

Hames, B. D. (1990) in “Gel Electrophoresis of Proteins,”

Hames, B. D., and Rickwood, D., eds., pp. 1–147, Oxford

University Press

Solubilization with

Laemmli buffer

SDS does not unfold some

proteases

Heat sample soon after adding LB

Avoids protein degradation by

proteases

1 pg of proteases degrades

proteins if not heated soon after

adding LB

Heating with Laemmli

buffer

Do not heat sample at 100 ˚C > 5

min

Asp-Pro bond most susceptible for

cleavage by heat and acidic

conditions

75˚C for 5 min avoids D-P bond

cleavage and inactivates proteases Volkin et al, 1995. Methods Mol. Biol. 40, 35–63

Deutsch, 1976. Anal Biochem 71, 300–303

Solubilizing

histones/membrane proteins

Heating with SDS LB alone may not

solubilize histones/memb proteins

6-8 M urea or Triton X-100

Insoluble material should be

removed by a 17,000 G spin (2 min)

This avoids streaking within gel

Protein aggregates in SDS

samples

Reductant becomes partly

oxidized, upon cooling sample,

part of the cysteines unprotected

Back-folding and creation of

inter-polypeptide aggregates

Blurred zones, formation of

double bands

Cooling sample to ~60 ˚C and

adding an alkylating agent

obviates this problem

Sharper bands

Artifacts abolished

Protein aggregates

(cont’d)

Galvani et al, Electrophoresis

2001, 22, 2066–2074

Miscalculating cross-

linking factor

Pore size of a polyacrylamide gel (a)

total concentration of acrylamide T

(b) degree of cross-linking C

Mistakenly assume that given total

acrylamide concentration T is the

percentage of acrylamide per volume

And C (crosslinking factor) is the

percentage of bis per volume Westermeier R. Practical Proteomics, 2007;

Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Miscalculating cross-

linking factor

a= mass of acrylamide in g

b= mass of bis in g

V= volume

Alternatively, use commercially available

acrylamide/bis stock solutions

Burgess, R.R. (2009) Methods Enzymol 463, 813-820. Review.

Electrophoresis

Overloading and underloading

commonly encountered

Overloading - distorted bands in

lane, also in adjacent lanes

Underloading - poor detection of

minor bands

Titrating running buffer in

SDS PAGE Use only Tris-base, glycine, and

SDS

Adjusting pH to 8.3 leads to

higher load of chloride

Longer protein separation times

Some zones remain poorly

resolved

SDS-PAGE

Best to polymerize gel O/N at RT

Haeberle gel - greatly accelerated

rate of polyacrylamide cross-linking

Run gel in 5 min, Haeberle RB at 70 ˚C

Haeberle J R. BioTechniques 23, 638-640, 1997

SDS-PAGE

Standard mini-gel, 2-4 hours

Pre-cast gel (4-20, 10%), in 10

min using heated Laemmli RB

Very recent results, showing

that electrophoresis, WB, and

immunoblotting - few hours

with actual run time of 60 min

Transfer buffers

Towbin buffer system (25 mM Tris,

192 mM glycine, 20% methanol ,

none to 0.01% SDS)

CAPS buffer system (CAPS, 10 %

methanol pH 11) for transfer to

PVDF prior to in situ blot

sequencing

Towbin transfer buffer

Transfer buffers without SDS are

better

Proteins can pass through

Immobilon-P membrane in the

presence of SDS

However, SDS needed (<0.01%)

during transfer of proteins that

precipitate

Methanol in transfer

buffer

Methanol aids in stripping SDS from

proteins

It stabilizes the geometry of the gel

binding capacity of NC for

protein

Helps proteins to bind better to NC

Methanol-less protein

transfer

Transferring high m. wt. proteins

Transferring conformation sensitive Ab

Enzyme activity needs to be

preserved

Drawback- PAGE gels swell in low-

ionic buffers

“bands” distorted if swelling occurs

during transfer

Blotting from SDS-PAGE

Proteins eluted as anions

Membrane placed on anode

side of gel

Remove bubbles bet NC and

gel, prevent bald spots

Gel +filter+ pads tightly held

together

Good transfer, no band

distortion

Do not exceed binding

capacity of membrane

Excess protein, weakly associated

with membrane, readily

accessible to antibody

Ensuing protein-antibody complex

wash off

Reduced signal

Inefficient transfer of high

m. wt. proteins

Reversible gel cross-linkers, gel

depolymerization

Limited protease digestion during

transfer

Prolonged transfer with addition

of SDS (0.1%)

Heat mediated transfer

Gershoni and Palade, Anal Biochem 131:1-15, 1983

Kurien and Scofield, J Immunol Methods 266:127-33, 2002

Preventing loss of low m.

wt. proteins

Low m. wt. proteins bind with low

affinities

Proteins lost during transfer or

washing

Cross-linking proteins to membrane

Use of 0.2 µm reduces “blow

through”

Conclusions

Simple method

Potential of western blotting

greatly evolved

Huge number of ways to transfer

proteins

Has led to a deeper

understanding of protein-ligand

interaction


Sponsored by:




Milan, Italy



Sciences Center

Oklahoma City, OK

Webinar Series

Nick Thomas, Ph.D.

GE Healthcare

Cardiff, Wales



July 30, 2014

Imagination at work

Nick Thomas PhD Principal Scientist GE Healthcare Life Sciences

Western Blotting The Art & The Science

Western Blotting Core technique in life science research

~37,000 labs worldwide ~60% of life sciences publications contain Western Blotting data Expression Characterisation Localisation Modification Diagnostics User intensive non-standardised workflow generates 35-45% variability in data Results depend on skill and experience with 25% failure rate

Pro

tein

Western Blotting Need for improvement

Standardisation and improved reproducibility

Better quantitation

Systematised simplified workflow

Get it right each and every time

Free up research time

“Did I put the stack in the blotter the

wrong way round ?”

“Looks like I forgot the

blocking buffer again !”

“####! – Can I run it again

tonight?”

“Well I thought

that old wash buffer was OK”

“What the #### ?!?!?”

Western Blotting From DAB to CCD

Started Western blotting with HRP colourimetric detection Applying photographic developing methods to Western blot processing First pre-stained molecular weight markers for SDS-PAGE and Western blotting Worked on development of Amersham™ ECL™ - the first commercially available chemiluminescence detection system

You don't take a photograph, you make it. Ansel Adams 1902-1984

Photographic Evolution Different science, same artistry

Western Blotting Evolution 20th to 21st century technologies

• HRP & Alk Phos • Nitrocellulose • Low sensitivity • Non-quantitative • Low reproducibility

• Range of detection substrates for higher sensitivity

• PVDF membranes • X-ray film and CCD

imagers • Quantitation

possible

• Quantitative • Standardisation to

HKP or total protein loading

• Multiplexing for 2-3 proteins and/or post-translational modifications

Colourimetric Chemiluminescent Fluorescent

Film Digital Imaging

Detection Improvements Dynamic range and multiplexing

Fluorescence Multiplexing Protein phosphorylation

Western Blotting Evolution Ready for the next stage

Integrate optimal technologies Systematise workflow Software integration for guiding, optimising and monitoring protocols to be; Standardised Quantitative Smart

Western Blotting Evolution Systems Integration

Streamline Workflows

Remove errors

Better artistry Better science

Western Blotting Evolution Different Science, Same Artistry

GE, imagination at work, and GE monogram are trademarks of trademarks of General Electric Company or one of its subsidiaries. Amersham, Cy, CyDye, ECL, ECL Advance, ECL DualVue, ECL Prime, ECL Plex, ECL Select, Hybond, and Rainbow are trademarks of GE Healthcare companies. ECL Advance contains Lumigen TMA-6 substrate and is sold under license from Lumigen, Inc. ECL Plus contains Lumigen PS3 substrate and is sold under license from Lumigen, Inc. ECL Prime: This product or portions thereof is manufactured and sold under license from Cyanagen Srl and is subject of US patent 7,855,287, US Patent 7,803,573, and Italian application number TO2010A000580, together with other equivalent granted patents and patent applications in other countries. CyDye: This product is manufactured under an exclusive license from Carnegie Mellon University and is covered by US patent numbers 5,569,587 and 5,627,027. The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes. A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare. Commercial use shall include: 1. Sale, lease, license or other transfer of the material or any material derived or produced from it. 2. Sale, lease, license or other grant of rights to use this material or any material derived or produced from it. 3. Use of this material to perform services for a fee for third parties, including contract research and drug screening. If you require a commercial license to use this material and do not have one, return this material unopened to GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-751 84 Uppsala, Sweden and any money paid for the material will be refunded. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.

©2008–2014 General Electric Company—All rights reserved. First published July 2014.

GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

www.gelifesciences.com/artofwesternblotting

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July 30, 2014



Milan, Italy



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Oklahoma City, OK

Nick Thomas, Ph.D.

GE Healthcare

Cardiff, Wales

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western blot tips and tricks - science | aaas slides_western... · twitter login (#sciencewebinar)...

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