PREPARING AND RUNNING SDS-PAGE GEL

Set the caster and gel casting plates

Prepare the resolvinggel.

Add the resolving gel between caster plates

Remove bubble from Resolving gel by adding 1ml isopropanol or Deionized water.

Wait for resolving gel to polymerize for 15-20 minutes

Pour the stacking gel

Prepare a Stacking Gel

Place a comb in the stacking Remove bubble from surface of stacking gel

Place the gel casting plates with gel in the electrophoresis tank

Allow gel to polymerize

Remove the gel casting gel plates along with gel from gel caster

Pour the tank buffer

Take out gel when sample runoff.

Load the prepared sample in the wells of stacking gel

Place the tank Lid Run the gel, at 60V initially when sample enter the resolving gel increase the volts to110V

Remove the comb from gel

Open the plates and remove gel from it in fixative solution

Stain the gel in coomassie brilliant blue R-250 dye

Destain the gel in destaining solution for about 24 hrs.

Visualize/imaging / quantification of proteins in the gel.

CASTING A SDS-PAGE GEL

Casting a SDS-PAGE gel

Place the plain plate (without notches) on the caster.

Open the Caster

Check the Leakage with Deionized water

Preparing 10ml, 12% Resolving gel

Allow to polymerize for 15 – 30 minutes.

Pour the gel between gel casting plates, till 2/3rd of the gel casting plates

Add 2.5 ml 1.5M Tris-cl (PH 8.8) Add 0.1 ml 10%SDS

solution

Add 1 ml isopropanol to remove bubble from face of resolving gel.

Add 0.004 ml TEMED

Add 0.1 ml 10%APS solution. (Freshly prepared)

Add 4.0 30% acrylamide mix. solution

Add 3.3ml Deionized water

Place a comb in the stacking gel between the gel casting plate.

Preparing a 5ml, 5% Stacking gel

Add 0.05 ml 10%SDS solution

Add 0.63 ml 1.0M Tris-cl (PH 6.8)

Add 0.83ml 30% acrylamide mix. solution

Add 3.4ml Deionized water

Remove the excessive isopropanol on the resolving gel and pour the stacking gel on the resolving gel.

Add 0.004ml TEMED

Add 40µl of BPB dye

Add 0.05 ml 10%APS solution

Allow to polymerize for 15-30 minutes

Place the plate with notches on it, and lock the caster gate.

Place the spacer on both extreme ends of the plate vertically

Place in PAGE tank and load sample and run the gel.

SAMPLE PREPARATION FOR SDS -PAGE

Preparation of dilution of sample in µg/µl in stock solution.

Preparation of different sample with different amount of protein in it.

Addition of water or SDS-PAGE loading dye or both.

Heating the sample at 95°C in hot water bath.

Loading the sample on SDS-PAGE gel.

BRADFORD ASSAY

Preparation of the assay reagent

Preparation of the dilutions of the given protein sample.

Preparation of the negative control (without protein sample) with water only.

Setting the wavelength of spectrophotometer to 595nm

Addition of 5ml of reagent to each dilution and wait for 5 minutes.

Taking OD of each dilution at 595nm and recording the results

Zeroing the spectrophotometer reading by using negative control.

Preparation of standard curve for estimation of proteins in sample.

SDS-PAGE GEL PRESERVATION

Drying at room temperature for about 24-36hrs.

Placing the SDS-PAGE gel on the membrane

Removing air bubbles between membrane and glass plate

Spreading the membrane on a glass plate

Soaking the nitrocellulose membrane in 20% glycerol for 40 minutes.

Removing air bubbles between two layers of membrane

Folding the membrane so that the gel is sandwiched in it.

Removing entrapped air bubbles between gel and membrane

MATERIALSMicropipettes (Blue, white, yellow and orange)

Micropipette tips (Blue, white and yellow)

1.5 ml Microcentrifuge Tubes

Falcon tubes

Test tube stands.

Centrifuge machine (7000-14000 rpm)

Spectrophotometer

Gel doc. Machine

SDS-PAGE apparatus (Wealtech or Biorad)

Gel Caster

Spacers

Comb of appropriate size.

Opener

Trays

Electric weight balance

Hot water bath

Icebox

Ice

Gloves

Aluminum foil

Deionized water

Distilled water

HPLC standard water

CHEMICALS/REAGENTS/BUFFERS 30% acrylamide mix. (29% Acrylamide + 1% Bisacrylamide) <!>

10% Sodium dodecyl sulfate

10% Amonium persulfate (prepared freshly)

TEMED (N,N, N´, N´ tetramethylethylenediamine)<!>

Tris-glycine Buffer

1.5M Tris-cl (PH 8.8)

1.0M Tris-cl (PH 6.8)

Coomassie Brilliant Blue R-250

Coomassie Brilliant Blue G-250

Glycerol

Butanol

Fixative Solution

Destain solution

2-Mercaptoethanol

95% Ethanol

Methanol

Acetic acid

32% HCl

85% phosphoric acid

Fermentas protein ladder (standard)

SODIUM DODECYL SULFATE POLYACRLYAMIDE GEL

ELECTROPHORESIS. (SDS-PAGE)

Principle of SDS-PAGESDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely

in biochemistry, forensics, genetics and molecular biology to separate proteins according to their

electrophoretic mobility. SDS gel electrophoresis of samples having identical charge per unit

mass due to binding of SDS results in fractionation by size and is probably the world's most

widely used biochemical method.

There are two kinds of gels which are used in the SDS-PAGE which are casted on each

other so that stacking gel is on the top of the resolving gel when the gel is casted. Stacking gel is

prepared and used with less percentage up to 5%. The main function of the stacking gel is to

stack all polypeptides of the protein sample loaded on gel on the face of the resolving gel. The

stacking gel is always casted on the resolving gel.

Resolving is prepared with variable percentages from 6-15% depending upon the size of

the protein loaded on the gel. The main function of the resolving gel is to give resolution of the

protein sample loaded according to its charge to mass ratio.

Chemical Ingredients of SDS-PAGE and their roles

Acrylamide <!>1) Polymerizes when dissolved in water

2) Neurotoxin in nature

Bisacrylamide <!>1) Helps in cross linking the gel molecule

2) A solution of acrylamide and bisacrylamide (29% and 1% respectively) is used to prepare

a PAGE gel.

SDS (sodium dodecyl sulfate)1) Detergent in properties and reduces surface tension.

2) Denature proteins

3) 10% solution of SDS is used in SDS-PAGE.

APS (ammonium per sulfate)1) provide free radical source

2) Freshly prepared 10% solution of APS is used in PAGE.

TEMED (N,N,N´,N´ tetramethylethylenediamine) <!>1) Catalyzes the polymerization of gel.

2) Highly carcinogenic in nature.

3) Light sensitive.

4) Usually TEMED is used in very low quantities.

Tris (tris aminomethane) Used as buffer in resolving gel, stacking gel and in tank buffer.

Glycine 1) source of slow ions

2) used as part of tank buffer

Chemicals/Reagents used in processing of SDS-PAGE gel and their

roleBromophenol Blue (BPB)

Bromophenol blue is a universal dye marker. BPB is mostly used as tracking dye and is

loaded on gel along with sample and it helps in tracking and stopping the gel before sample

runoff the gel.

Coomassie Briliant Blue R-250 (CBB)CBB is most popular protein stain, which binds the protein non-specifically. It is used in

the staining the gels and excess dye is incorporated in the protein and gel can be destained.

Blue bands of proteins can be observed with white back ground when destained.

Glycerol It is preservative and a weightening agent. Addition of the glycerol is recommended for

storage of enzymes. Is also helps to weigh down the sample into the wells without being spread

while loading

Butanol Water saturated butanol is used as an overlay solution on resolving gel.

2-Mercaptoethanol It is a reducing agent used to disrupt disulfide bonds to ensure protein is fully denatured

before loading on gel; ensuring the proteins run uniformly.

Solution preparation for SDS-PAGE

30% acrylamide mixture (100ml)Mixed 29gm of acrylamide and 1gm of bisacrylamide and made volume up to 100ml

with deionized water. The solution was stored at 4°C in dark bottle.

Precaution for solution preparation

Always wear gloves and use mask while making acrylamide mixture solution because it

is highly neurotoxic compound and can cause problem in inhaling.

10% SDS solution (100ml)Took 10gm of sodium dodecyl sulfate and made final volume up to 100ml with deionized

water, dissolved and stored at room temperature.

1.5M Tris-Cl (pH 8.8) resolving buffer

Dissolved 36.3 of tris base in 150ml of deionized water and adjusted pH to 8.8 with 32%

HCl and made final volume up to 200ml. Stored at 4°C.

1.0M Tris-cl (pH6.8) stacking buffer (100ml)Dissolved 12.1gm of tris base in 70 ml of deionized water, and adjusted pH to 6.8 by

using 32% Hcl and 1M sodium hydroxide solution and stored at 4°C.

10% APS solution (1ml)Always prepared freshly

Dissolved 0.1gm of APS in 1ml of deionized water and stored at room temperature.

Fixative solution (1000ml)10% acetic acid = 100ml

30% ethanol = 300ml

Final volume made up to 1000ml with distilled water.

Coomassie Brilliant Blue R-250 (100ml) [staining solution]Dissolved

Coomassie Brilliant Blue R-250 = 0.25 g

Methanol : H2O

1 : 1

45ml : 45ml = 90 ml

Glacial acetic acid = 10ml

Stored at room temperature.

Sample loading buffer 3X stock (10ml)Dissolved

1M Tris-Cl pH 6.8 2.4 ml

20% SDS 3 ml

Glycerol (100%) 3 ml

2-mercaptoethanol 1.6 ml

Bromophenol blue 0.006g

stored 4°C

5X Tris-Glycine tank buffer (1000ml)Dissolved

15.1 g of Tris-base

94 g of glycine

50ml of 10% SDS solution

Made final volume up to 1000ml with distilled water.

Destaining Solution (1000ml)Dissolved

50ml of methanol

70ml of acetic acid

Made final volume up to 1000ml with distilled water.

PROTOCOL FOR SDS-PAGESetting up gel caster:

1) Opened the gel caster.

2) Washed the gel caster plates with 70% alcohol and air dried.

3) Placed the plain plate without notches in the gel caster.

4) Placed the spacers on each side of gel caster plate.

5) Placed the gel caster plate with notches in the gel caster and closed the door of the gel

caster.

6) Checked the leakage by adding deionized.

Gel caster opened

Gel caster closed

Preparing and pouring 12% and 10ml resolving gel:1) Took a falcon tube.

2) Added 3.3 ml of deionized water.

3) Added 4.0ml of 30% acrylamide mixture.

4) Added 2.5ml of 1.5MTris-cl buffer (pH8.8).

5) Added 100µl of 10% SDS.

6) Added 100µl of 10% freshly prepared APS.

7) Added 4µl of TEMED.

*adding 1.5 times more volume of TEMED can reduce the time of polymerization of gel.

8) Poured the gel with the help of 1ml micropipette until 2/3rd of the gel casting plates.

9) Quickly added 1ml of isopropanol to the gel before the polymerization of the gel. This

will remove bubbles from the face of resolving.

Gel caster door

10) Waited for 15-20minutes until the gel polymerized.

Preparing and pouring 5% and 5ml stacking gel:1) Took a fresh falcon tube.

2) Added 3.4ml of water

3) Added 0.83ml of 30% acrylamide mixture.

4) Added 0.63ml of 1.0MTris-cl (pH 6.8).

5) Added 50µl of 10% SDS solution.

6) Added 40µl of BPB dye.

7) Added 50µl of 10% freshly prepared APS.

8) Added 4µl of TEMED.

9) Mixed, and quickly poured the stacking gel in the gel casting plates.

10) Pulled the bubbles aside with comb and placed the comb between the plates before the

polymerization of the gel.

11) Waited for 10-15 minutes until the gel polymerized.

Sample preparation for SDS – PAGE Prepared different concentration of Human blood plasma in terms of µg/µl in deionized water.

Added 1X BPB SDS-PAGE loading dye to the sample and heated at 95°C in a water bath for 5

minutes.

Running SDS-PAGE1) Opened the gel caster and took out gel casting plates with gel intact.

2) Placed the gel caster plates in the PAGE apparatus and tighten it.

3) Poured 1x tris-glycine tank buffer in the PAGE apparatus tank until the plates sank in it.

4) Took out the comb from the gel.

5) Took sample and loaded 10µl of it in the wells of the gel.

Power supply PAGE tank

Initial run of SDS- PAGE change in volts when sample entered in resolving gel

6) Run the electrophoresis at 60V initially, when sample run off the stacking gel the

voltages were increased to 110V. The process was allowed to run about 1-2.5 hours.

7) When sample ran off the resolving gel, stopped the

voltages and took out the gel casting plates with gel

intact.

8) Opened the gel casting plates with the help of an

opener, and separate out the gel by shaking the gel in the

fixative solution in a tray.

9) Transferred the gel to another tray and added coomassie brilliant blue so that the gel was

dipped in it. Placed the tray on the shaker for 5 minutes.

Stained gel Destained gel

10) Removed the excessive dye with the help of a dropper.

11) Added destain solution to the tray and placed it on the shaker for 10 minutes.

12) Replaced the destain solution with fresh one and left the gel for destaining over night.

13) After 24 hrs observed the gel.

14) Blue bands of protein were observed with white background after destaining.

15) Took digital image of gel with help of gel doc. using white light.

Protein bands

Running samplePAGE tank

UPS

Precautions:1) Always check the leakage of gel caster plate, before adding the resolving gel.

2) Remove bubble from the face of the stacking gel, and make sure that there is no bubble

left in the wells. This can interfere with protein resolution.

3) Always wear gloves while handling the TEMED.

4) Keep all the samples on ice before loading.

5) Do not heat the sample more than 5 minutes.

6) Wear gloves while handling the gel.

7) Do not stain the gel more than 5 minutes, this will over stain the gel and can delay results

of the experiment.

8) SDS – PAGE gel is very delicate, be very careful while handling the gel.