Western blot | Gel Electrophoresis

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

Gel Electrophoresis

1. Preparation of Gel:

Part-1:

• Clean glass plates with D.W and 70% ETOH, dry thoroughly and assemble them.
• Place 100% ETOH between glass plates to check for leakage.
• If the ETOH does not leak, clean the ETOH using 3MM paper.
• Carefully insert the comb, mark it in the comb down to 0.5 cm, and remove the comb.

Part-2:

• Identify the molecular weight of the protein to be separated and determine the concentration of acrylamide.

Table-1: Acrylamide concentration as per protein size.

Acrylamide concentration (%)	Protein size (kDa)
15	10-43
12	12-60
10	20-80
7.5	36-94
5	57-212

Table-2: Preparation of running gel.

Running Gel (10 ml)	5％	7.5％	10％	12％	15％
D.W H2O	5.3 mL	4.6 mL	4 mL	3.3 mL	2.3 mL
1.5 M Tris-Cl (pH 8.8)	2.5 mL	2.5 mL	2.5 mL	2.5 mL	2.5 mL
30 acrylamide Solution	2 mL	2.7 mL	3.3 mL	4 mL	5 mL
10 SDS	100 µL	100 µL	100 µL	100 µL	100 µL
TEMED	8 µL	6 µL	4 µL	4 µL	4 µL
10 APS	100 µL	100 µL	100 µL	100 µL	100 µL

Note: Before adding APS and TEMED to the solution, thoroughly mix the solution, settle it down for 5-10 mins and after then add APS and TEMED and mix the solution very gently to avoid any air bubble.

• Running Gel is poured to the bottom of the comb to a depth of about 0.5 cm.
• Carefully put 1 ml of Isopropanol (or 1 ml of water-saturated N-butanol) and let the gel dry.
• After 30-40 minutes, remove the Isopropanol and wash with D.W.
• After wiping with 3MM paper, re-mark the position of the running gel (let gel down a lot).

Table-3: Preparation of stacking gel.

5% Stacking gel	2ml	4ml	6ml	8ml
D.W H2O	1.4 mL	2.7 mL	4.1 mL	5.5 mL
1.5 M Tris-Cl (pH 6.8)	250 µL	500 µL	750 µL	1000 µL
30 acrylamide Solution	330 µL	670 µL	1000 µL	1300 µL
10 SDS	20 µL	40 µL	60 µL	80 µL
TEMED	2 µL	4 µL	6 µL	8 µL
10 APS	20 µL	40 µL	60 µL	80 µL

Note: Before adding APS and TEMED to the solution, thoroughly mix the solution, settle it down for 5-10 mins and after then add APS and TEMED and mix the solution very gently to avoid any air bubble.

• Pour 5% stacking gel and carefully insert the comb so the bubble does not get in.
• If you combine a stacking gel in a comb, you will get less bubble.
• After inserting the comb, fill the stacking gel in the space where the stacking gel is not enough so that the well does not collapse.
• After 30-60 minutes, if the stacking gel hardens, remove the comb carefully and clean the wells with a syringe to make it easier to load the protein into the wells.
• Transfer the small glass into the electrophoresis tank with the inside facing, then fill the 1X SDS running buffer so that it does not leak.

B. Sample preparation & loading:

1. While the gel is firm, pre-quantify the protein sample (stored at -80 ° C) in ice and dissolve.
2. Set the temperature of the dri-bath to 95 ° C in advance.
3. Mix the same amount of 2X sample buffer with the protein sample.
4. Heating with above samples at 95 ° C for 10 min [Protein denaturation]
5. Cooling at ice (4 ℃) for 10 min.
6. Spin-down lightly, then load the gel and store the remaining sample at -80 ° C.

Table-4: Special recommendation for sample preparation.

Ingredients	Quantity	Recommendation
Marker	3-4 microL	Do not vortexing
Sample+2x Sample buffer	10 ul + 10 ul	Pipetting
Loading dye	10 ul (the same amount as the sample)	Heat block with 95 ° C for 10 min

C. Running the gel:

• Stacking gel: 80-100 V (mainly 80 V to 20 min)
• Running gel: 120V (about 1.5 hr) → run until the dye reaches the bottom of the gel, usually 20 minutes after running out of dye.