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14 Polycrylamide-Sodium Dodecyl Sulphate Slab Gel Electrophoresis (SDS-PAGE) of Proteins (Laemmli 1970)

14 Polycrylamide-Sodium Dodecyl Sulphate Slab Gel Electrophoresis (SDS-PAGE) of Proteins (Laemmli 1970)

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116



7



Qualitative and Quantitative Estimations of Amino Acids and Proteins



Principle

SDS is an anionic detergent which binds strongly to, and denatures, proteins.

The number of SDS molecules bound to a polypeptide chain is approximately

half the number of amino acid residues in that chain. The protein–SDS complex

carries net negative charges, hence moves towards the anode and the separation is

based on the size of the protein (charge to mass ratio).

Reagents

1. Stock acrylamide solution

Acrylamide 30% – 30 g

Bisacrylamide 0.8% – 0.8 g

DD Water to – 100 mL

2. Separating gel buffer

1.875 M Tris–HCl – 22.7 g (pH 8.8)

Water to – 100 mL

Stacking gel buffer

0.6 M Tris–HCl – 7.26 g (pH 6.8)

Water to – 100 mL

3. Polymerising agents

(a) Ammonium – 0.5 g/10 mL, prepare fresh before use persulphate 5%

(b) TEMED – fresh from the refrigerator

4. Electrode buffer (may be used 2–3 times)

0.05 M Tris – 12 g

0.192 M Glycine – 28.8 g (pH 8.2–8.4)

0.1% SDS – 2 g

Water to – 2 L

5. Sample buffer (5Â concentration)

Tris–HCl Buffer, pH 6.8 – 5 mL

SDS – 0.5 g

Sucrose – 5 g

Mercaptoethanol – 0.25 mL

Bromophenol Blue – 1 mL

(0.5% W/V solution in water)

Water to – 10 mL

Dilute to 1Â concentration and use. Store frozen in small aliquots.

6. Sodium dodecyl sulphate 10% solution – store at room temperature and use



7.14



Polycrylamide-Sodium Dodecyl Sulphate Slab Gel Electrophoresis. . .



117



7. Standard marker proteins

Protein



MW (Daltons)



a-Lactalbumin

Trypsin inhibitor soybean

Trypsinogen

Carbonic anhydrase

Glyceraldehydes-3-phosphate dehydrogenase, rabbit

Albumin, egg

Albumin bovine



14,200

20,100

24,000

29,000

36,000

45,000

66,000



Dissolve the above proteins in single strength sample buffer at a concentration each to 1 mg/mL. Load the well with 25–50 mL.

8. Protein stain solution

Coomassie Brilliant Blue R250 – 0.1 g

Methanol – 40 mL

Acetic acid – 10 mL

Water – 50 mL

Dissolve the dye in methanol first and use fresh preparation every time.

9. Destainer

As for staining solution but without dye.

Procedure

1. Thoroughly clean and dry the glass plates and spacers, then assemble them

properly. Hold the assembly together with clips. Clamp in an upright position.

White petroleum jelly or 2% agar (melted in a boiling water bath) is then applied

around the edges of the spacers to hold them in place and seal the chamber

between the glass plates.

2. Prepare a sufficient volume of separating gel mixture (30 mL) for a chamber of

about 18 Â 9 Â 0.1 cm by mixing the following:

Stock acrylamide solution (mL)

Tris–HCl, pH 8.9 (mL)

Water (mL)

Degas the solution for 3–5 min

and then add

Ammonium persulphate

solution (mL)

10% SDS (mL)

TEMED (mL)



For 15% gel



For 10% gel



20

8

11.4



13.3

8

18.1



0.2



0.2



0.4

20



0.4

20



Mix gently and pour the gel solution in the chamber between the glass plates.

Layer distilled water on top of the gel and leave to set for 30–60 min.



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Qualitative and Quantitative Estimations of Amino Acids and Proteins



3. Prepare stacking gel (4%) by mixing the following solutions (total volume

10 mL).

Stock acrylamide solution ¼ 1.35 mL

Tris–HCl (pH 6.8) ¼ 1 mL

Water ¼ 7.5 mL

Degas as above, and then add:

Ammonium persulphate solution (5%) ¼ 50 mL

10% SDS ¼ 0.1 mL

TEMED ¼ 10 mL



4.



5.



6.



7.



8.



9.



Remove the water from the top of the gel and wash with a little stacking gel

solution. Pour the stacking gel mixture, place the comb in the stacking gel and

allow the gel to set (30–60 min).

After the stacking gel has polymerized, remove the comb carefully without

distorting the shapes of the well. Install the gel after removing the clips, agar,

etc. in the electrophoresis apparatus. Fill it with electrode buffer and remove

any trapped air bubbles at the bottom of the gel. Connect the cathode at the top

and turn on the DC-power briefly to check the electrical circuit. The electrode

buffer and the plates can be kept cooled using cooling facility so that heat

generated during the run is dissipated and does not affect the gel and

resolution.

Prepare samples for electrophoresis, following suitable extraction procedure.

Adjust the protein concentration in each sample using the 5-strength sample

buffer and water in such a way that the same amount of protein is present per unit

volume. Again the concentration should be such as to give a sufficient amount of

protein (50–200 mg) in a volume (25–50 mL) not greater than the size of the

sample well. Generally, the sample solution is heated in boiling water for

2–3 min to ensure complete interaction between proteins and SDS.

Cool the sample solutions and the required volume is filled in a microsyringe and

injected into sample well through the electrode buffer. Marking the position of

wells on the glass plate with a marker pen and the presence of bromophenol blue in

the samples, also load in to a well a standard marker proteins in the sample buffer.

Turn on the current to 10–15 mA for initial 10–15 min until the samples travel

through the stacking gel. The stacking gel helps concentration of the samples.

Then continue the run at 30 mA until the bromophenol blue reaches the bottom

of the gel (about 3 h). However, the gel may be run at a high current (60–70 mA)

for short period (1 h) with proper cooling.

After the run is compete, carefully remove the gel from between the plates and

immerse in staining solution for at least 3–4 h or overnight with uniform

shaking. The proteins absorb the Coomassie Brilliant Blue.

Transfer the gel to a suitable container with at least 200–300 mL destaining

solution and shake gently and continuously. Dye that is not bound to proteins is

removed. Change the destainer frequently, particularly during initial periods,

until the background of the gel is colourless. The proteins fractionated into band

are seen coloured blue. As the proteins of minute quantities are stained faintly,



7.15



Fluorography of Polyacrylamide Gels



119



destaining process should be stopped at appropriate stage to visualize as many

bonds as possible. The gel can now be photographed or dried in vacuo for a

permanent record.

Precautions

1. Acrylamide as a monomer is highly neurotoxic; handling should be with

extreme care.

2. All solutions should be prepared fresh, old solutions result in poor resolution of

proteins.

3. All chemicals and distilled water should be of high quality. The solution

prepared should be filtered before use. The solution can be stored refrigerated

for 1–2 weeks.

4. Prefer to use the gel immediately following polymerization although the

separation gel after setting can be stored overnight by wetting with fourfold

diluted separation gel buffer or with stacking gel and comb placed over it to

avoid drying.

5. Degassing of gel mix should be adequate for easy polymerization.

6. The water layered over the separation gel should be completely removed for

quick polymerization of the stacking gel.

7. Handle the polyacrylamide gel carefully to avoid any breakage.

8. The slab gel along the glass plates is placed vertically in the electrophoresis

tank and run. It is therefore called “vertical slab gel electrophoresis”.

9. In 10% polyacrylamide gels, the low molecular weight (~10,000 daltons)

polypeptides will migrate diffused; for fine resolution of these polypeptides

use gels of higher (15%) acrylamide concentration.

10. Any band of 0.1 mg protein is visualized by Coomassie Brilliant Blue staining

in SDS-PAGE; for visualizing proteins of lower concentration below 0.1 mg

high sensitive (silver staining) method is recommended (Table 7.4).



7.15



Fluorography of Polyacrylamide Gels

(Bonner and Laskey 1974)



Fluorography is an improved version of autoradiography in the presence of a

fluorescing compound. It is one of the technique to determine the radioactivity in

gels and other media by a combination of fluorescence and photography. When

radioactivity labelled macromolecules such as proteins are separated by electrophoresis much of the radiation is absorbed by the gel and the autoradiography of

such gel gives poor results. For low-energy emitters such as tritium and 14C

the sensitivity of fluorography is many-fold higher than that of autoradiography.

The fluorography may be photographed for qualitative information or analysed by

scanning for quantitative results.



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Qualitative and Quantitative Estimations of Amino Acids and Proteins



Table 7.4 Frequent troubleshooting and remedies during electrophoretic procedure

Trouble

Cause

Remedy

Failure or slow

polymerization

of the gel



Presence of oxygen,

Absence of catalysts

Stock solution aged

Glass plates

Poor sample wells

Stacking gel and comb

Long duration of the run Interference of air-bubbles

Staining is poor

The dye absorption not

efficient

The staining is patchy

Solid dye

The stained bands are

The dye is removed

decolourised

excessively

Protein bands are in

Insufficient electrophoresis

adequately resolved

separation gel

Protein bands wavy

Excess persulphate

Bands have become

Proteins remain

streaked

aggregated, denatured

or insoluble

Protein dye migration

Gel is partly insulated by

not even

air bubbles+

insufficient cooling

The protein bandlane

Sample density

broadens at the

bottom of separation

gel

Sample diffuses while

Low density of sample

loading the wells



Degas the solution efficiently

Check if all solutions mixed

Use fresh solutions

Degrease the plates with ethanol

Fit and or remove the comb carefully

Flush air-bubbles

The dye may be old, use a strong dye

solution

Dissolve the dye completely or filter

Restain the gel and stop destaining

appropriately

Run for longer time. Change the

percentage of the gel

Use optimum concentration of persulphate

Use fresh sample buffer or extra SDS or

centrifuge the sample extract

sufficiently

Remove air bubbles before

electrophoresis improve the cooling or

run at a low current

Load equal volume of sample in each well,

equal strength sample buffer, leave no

empty well in the middle

Increase the concentration of sucrose/

glycerol in the sample buffer



Principle

In fluorographic technique, a fluro/scintillator impregnated into the gel absorbs the

radiation from the isotope and re-emits light that passes through the gel to the film

producing a photographic image analogous to an autoradiograph.

Materials

1. Dimethyl sulphoxide (DMSO)

2. PPO (2,5 dipheynl oxazole) solution: Dissolve 22 g PPO and make up to 100 mL

in DMSO

3. Fixing solution: 7% acetic acid, 20% methanol in distilled water.

4. Film developer

5. Photographic flash unit (with red screen on the flash window)

6. X-ray film

7. Gel dryer



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