Methods and Protocols for Western Blot

Overview of the Western Blot

The western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein, and to measure the amount of protein expression. 

Step 1: Preparation and quantification of protein samples

1.     Cell or tissue protein extraction

2.     Protein concentration measurement (BCA method)

l  Standard Hole: add ultrapure water according to the instructions, then add standard protein solution

l  Target Hole: add 18µl ultrapure water + 2µl target protein

l  Prepare BCA fluid according to the instructions, add 200ul per hole

l  Measure the concentration after placing it at 37℃ for half an hour

3.     Calculate the sample amount and design the sample order

l  Sample volume: sample volume/concentration x 5/4

l  Sample order: no treatment, control, treatment;

Tips: The volume should not be too different.

Step 2: Electrophoresis

1.     Clean the glass plate, rinse with deionized water, air-dry or bake

2.     Mix glue according to the instructions. Separating glue and Concentrated glue

3.     Preparation of electrophoresis solution (tris 3.03g, glycine 14.4g, SDS1g, 1L)

4.     Put the glass plate in the electrophoresis tank, add electrophoresis solution, and gently pull out the comb

5.     Add sample

6.     Connect the power supply, set the voltage and time. Concentrated glue (90v, about 40min, the maker separates the strip), change the voltage, separating glue (120v, 2h). (Don't let bromophenol blue go out)

Step 3: Transfer

1.     Preparation of electro-transfer solution (tris3.03g, glycine 14.4g, methanol 100ml, 1L)

2.     Peel glue, cut glue, cut membrane, and activate membrane in the electro-transfer solution (methanol 1min, deionized water 1min, electrolyte 15min)

3.     "Sandwich" blackboard-sponge-filter paper-glue-membrane- filter paper-sponge~white board, clamp it and put it into the electro-rotor tank.

4.     Connect the power supply, constant current and time.

Schematic of western blot transfer of proteins from a polyacrylamide gel to a membrane.

Step 4: Western Blot

1.     Membrane treatment (methanol 1min, air-dry, methanol 1min, water 1min)

2.     Closed (TBST with 5% milk), 1 hour with shaker

3.     Add the primary antibody, dilute with milk

4.     Incubate at room temperature for 20min

5.     Wash with 0.1%TBST, 10min x 3 with shaker

6.     Add the secondary antibody, dilute with milk, 1 hour with shaker

7.     Wash with 0.1%TBST, 10min x 3 with shaker

Step 5: Result Analysis

1.     Smile band

l  Analysis: The gel solidified uniformly, which usually occurs in thicker gels

l  Solution: Mix the glue thoroughly, do subsequent tests after it fully solidified

2.     Texture phenomenon

l  Analysis: Sample insoluble particles

l  Solution: Centrifuge the sample before adding the sample

3.     Thick electrophoresis band

l  Analysis: The protein sample is not concentrated and the amount is large

4.     There are many holes in the band

l  Analysis: The bubbles were not removed in the “sandwich”

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