Western blot analysis to confirm the protein expression

Western blot analysis to confirm the protein expression

A western blot defined as the electrophoresis of the antigen followed by its subsequent transfer to nitrocellulose paper and incubation with specific antibody and then with labelled secondary antibody.

Procedure:

The protein samples will be prepared by extraction the leaf samples with protein buffer (Disodium hydrogen phosphate (Na₂HPO₄) 2.38 g, Potassium dihydrogen phosphate (KH₂PO4) 0.40g, Potassium chloride (KCl) 0.40g, Sodium chloride (NaCl),16.00 g, Distilled water 2.00 lt, pH 7.40) and separated by SDS-PAGE.

Clean and dry the glass plates and spacers, then assemble them properly. De-gas the resolving gel solution (18.15 g Tris-base dissolved in 80.0 ml distilled water, pH adjusted to 8.8 with I N HCI, volume made to 100 ml with distilled water and stored at 4° C.) using a vacuum for 3-5 min and then add ammonium persulphate solution.

Mix gently and carefully, pour the gel solution in the chamber between the glass plates, leaving the space for stacking gel. Layer distilled water or isobutanol on the top of the gel and leave to set for ~ 15 min.

Degas the stacking gel solution (12.0 g Tris-base dissolved in 60 ml distilled water, pH adjusted to 6.8 with I N HCI, volume made to 100 ml with distilled water and stored at 4° C.) and then add ammonium persulphate solution.

Remove the water from the top of the gel. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set.

After the stacking gel has polymerized, remove the comb without distorting the shapes of the well. Wash the wells with distilled water using a syringe.

Prepare samples for electrophoresis. Adjust the protein concentration in each sample so that the same amount is present per unit volume.Again the concentration should be in the range of 20-200 µg protein in a volume (about 50 µL) not greater than the size of the sample well.

Resolving (Separating) gel composition (12%) (10ml) Distilled water 3.3, 30% Acrylamide mixture 4.0 mL, 1.5M tris (pH 8.8) 2.5 mL, 10%SDS 0.1 mL, 10%APS 0.1 mL, TEMED 0.004 mL, prepared just before use by dissolving 100 mg in 1 ml distilled water.

The first four solutions mixed in a beaker and then solutions 5 and 6 added by swirling the beaker gently to mix and quickly transferred to a gel mould.

Stacking gel preparation (5%) 4mL- Distilled water 2.7mL, 30% Acrylamide mixture 0.67 mL, 1 M Tris (pH 6.8) 0.5 mL, 10% SDS 0.04 mL, 10% APS 0.004 mL, TEMED 0.004 mL.

The first four solutions mixed in a beaker and then solutions 5 and 6 added by swirling the beaker gently to mix and quickly transferred to a gel mould. Fill the upper and lower tank with electrode buffer (Tris-base 15.0 g, Glycine-72.0 g, SDS-5.0 g, Dissolved in distilled water, pH adjusted to 8.3, volume made up to 1000 ml with distilled water and stored at 4° C. The buffer warmed to 37° C before use.

This buffer diluted at 1:4 (v/v) with distilled water) and connect upper trough to cathode (-) and lower trough to anode (+). Turn on the current to 10-15 mA for initial 10-20 min until the sample travel through the stacking gel. Then continue the run at 30 mA until the bromophenol blue reaches the bottom of the gel (about 3 hours).

After the electrophoresis carefully remove the gel from between the glass plates and immerse in the staining solution (Coomassie blue G-250 - 0.08 g Fixing solution - 200 mL).

Destain the gel with several changes of destaining solution (Glacial acetic acid- 30 mL, Conc. Methanol- 60 mL, DDW- 510 mL) till the background becomes clear.

Blotting to Nitrocellulose membrane

Once SDS-PAGE is ready, Whatmann no 1 filter paper cut to the same dimension as that of the gel. Usually 12 such pieces cut out and presoaked in the transfer buffer (0.025 M Tris- Glycine buffer, pH 8.8 and 0.1% SDS). The nitro cellulose membrane was also cut exactly to the same size as that of the gel and soaked in transfer buffer prior to use.

The gel will be soaked in the transfer buffer for equilibration. Preparation of the transfer stack: 6 pieces of filter paper were stacked over one another followed by the nitrocellulose membrane, gel and over the gel another 6 pieces of filter paper were placed one over the other.

Before placing the gel, suitable marks are made to indicate the position of the marker lane. Eletroblotting should be done for 2 hrs at 50 volts and 45mA current. The membrane is kept at 4°C overnight.

The membrane is then placed in the blocking solution for 1Hr . (The blocking is done so that the area of the nitrocellulose where no protein has been transferred is occupied by milk powder, which in turn prevents non-specific binding of the antibody).

After 1 hr blocking solution was poured out and washed the nitrocellulose membrane with PBST wash solution. 3 washes of 10 min each should be done.

The nitrocellulose membrane will be placed in the primary antibody solution and kept at room temperature on a rocker for 1 hr.

The membrane will be washed with PSBST wash solution. 3 washes of 10 min each will be carried out, changing the wash solution after each wash. After washing, the membrane will be placed in the secondary antibody solution for 1 hr at room temperature on a rocker.

Later the membrane will be washed with PSBT wash solution. 3 Washes of 10 min each will be carried out, changing the wash solution after each wash. After washing, the membrane was placed in substrate solution which provides colour only where there is binding of the primary and secondary anti body.

Usually colour develops within 30 min. The reaction was carried out in dark conditions. The reaction will be stopped by rinsing the membrane with water and dried under dark conditions. The membrane should not be exposed to light as the bands fade out. Photograph will be taken for permanent record.

	The protein samples will be prepared by extraction the leaf samples with protein buffer (Disodium hydrogen phosphate (Na₂HPO₄) 2.38 g, Potassium dihydrogen phosphate (KH₂PO4) 0.40g, Potassium chloride (KCl) 0.40g, Sodium chloride (NaCl),16.00 g, Distilled water 2.00 lt, pH 7.40) and separated by SDS-PAGE.
	Clean and dry the glass plates and spacers, then assemble them properly. De-gas the resolving gel solution (18.15 g Tris-base dissolved in 80.0 ml distilled water, pH adjusted to 8.8 with I N HCI, volume made to 100 ml with distilled water and stored at 4° C.) using a vacuum for 3-5 min and then add ammonium persulphate solution.
	Mix gently and carefully, pour the gel solution in the chamber between the glass plates, leaving the space for stacking gel. Layer distilled water or isobutanol on the top of the gel and leave to set for ~ 15 min.
	Degas the stacking gel solution (12.0 g Tris-base dissolved in 60 ml distilled water, pH adjusted to 6.8 with I N HCI, volume made to 100 ml with distilled water and stored at 4° C.) and then add ammonium persulphate solution.
	Remove the water from the top of the gel. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set.
	After the stacking gel has polymerized, remove the comb without distorting the shapes of the well. Wash the wells with distilled water using a syringe.
	Prepare samples for electrophoresis. Adjust the protein concentration in each sample so that the same amount is present per unit volume.Again the concentration should be in the range of 20-200 µg protein in a volume (about 50 µL) not greater than the size of the sample well.

	Once SDS-PAGE is ready, Whatmann no 1 filter paper cut to the same dimension as that of the gel. Usually 12 such pieces cut out and presoaked in the transfer buffer (0.025 M Tris- Glycine buffer, pH 8.8 and 0.1% SDS). The nitro cellulose membrane was also cut exactly to the same size as that of the gel and soaked in transfer buffer prior to use.
	The gel will be soaked in the transfer buffer for equilibration. Preparation of the transfer stack: 6 pieces of filter paper were stacked over one another followed by the nitrocellulose membrane, gel and over the gel another 6 pieces of filter paper were placed one over the other.
	Before placing the gel, suitable marks are made to indicate the position of the marker lane. Eletroblotting should be done for 2 hrs at 50 volts and 45mA current. The membrane is kept at 4°C overnight.
	The membrane is then placed in the blocking solution for 1Hr . (The blocking is done so that the area of the nitrocellulose where no protein has been transferred is occupied by milk powder, which in turn prevents non-specific binding of the antibody).
	After 1 hr blocking solution was poured out and washed the nitrocellulose membrane with PBST wash solution. 3 washes of 10 min each should be done.
	The nitrocellulose membrane will be placed in the primary antibody solution and kept at room temperature on a rocker for 1 hr.
	The membrane will be washed with PSBST wash solution. 3 washes of 10 min each will be carried out, changing the wash solution after each wash. After washing, the membrane will be placed in the secondary antibody solution for 1 hr at room temperature on a rocker.
	Later the membrane will be washed with PSBT wash solution. 3 Washes of 10 min each will be carried out, changing the wash solution after each wash. After washing, the membrane was placed in substrate solution which provides colour only where there is binding of the primary and secondary anti body.