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Location: Home > Information Center > Technical FAQs > ELISA Kit Technology Column > Western Blot Proteins Sample Preparation

Western Blot Proteins Sample Preparation

Date: 2015-04-10 Author: Leading Biology Click: 4815

Western Blot Proteins Sample Preparation

Before doing western blot, it’s crucial to extract the proteins. It almost contributes to both success and failure. Now let’s start:


Extract the total protein of the monolayer adherent cells:

1. Pour the culture solution and upend the bottle on the absorbent paper to remove the liquid left (or place the bottle upright for a while and blot the liquid in the bottom with a pipettor).

2. Add 3 ml 4℃ pre-cooling PBS to each bottle of cells (0.01 M pH 7.2~7.3). Keep it flat and Shake slowly for 1 min to wash the cells and then throw the washing liquor away. Repeat the operation above for 2 times (3 times in total) to wash the culture solution away. Remove PBS thoroughly and put the culture bottles on the ice.

3. Add 10 μl PMSF (100 mM) into 1 ml lysate and place them on the ice after shake well. (Shake the PMSF until no crystal left before mix with lysate)

4. Add 400 μl lysate containing PMSF to each bottle of cells, and lyse on the ice for 30 min. To lyse the cells thoroughly, the bottles should be shaken back and forth often.

5. After lysis, scrape the cells to the other side of the bottle with a clean stick (Be quick), and then transfer the cell debris and lysate to a 1.5 ml centrifuge tube with a pipettor (Operate on the ice is preferred).

6. Centrifuge them under the condition 4℃、12000 rpm for 5 min.(Pre-cool the centrifuge)

7. Supernate subpackage in 0.5 ml centrifuge tubes and preserve at -20℃.

(In my opinion, the operability of the methods above needs to be improved further. The proteins in a cell is very little. Sometimes a bottle of 50 ml cells could only add 100-200μl lysate according to the experimental requirements. In the light of the operation above, it’s insufficient to adhere to the bottle wall with 200μl lysate. Here is an operable method. Add pre-cooling PBS for several milliliters, then scrape the cells and transfer to test tubes. If we collect one type of protein from several bottles, we could put it in one tube and transfer to eppendorf tubes after centrifuge)


Extract the total protein of the tissues:

1. Put a small amount of tissue block in the spherical part of an 1-2 ml homogenizer. Cut it with clean scissors as small as possible.

2. Add 400 μl single detergent lysate (containing PMSF) in a homogenizer and pestle, then place it on the ice.

3. Pestle for a while and put it on the ice after a few minutes. Repeat for several times to make the tissue fine crushing.

4. After lysis for 30 min, transfer the lysate to an 1.5 ml centifuge tube with a pipettor and centrifuge under the condition 4℃、12000 rpm for 5 min. Supernate subpackage in 0.5 ml centrifuge tubes and preserve at -20℃.


Extract the total protein of adherent cells treated with drugs

Because of the effect of the drugs, some cells fall down. So we should collect the cells in the culture solution in addition. The following is the method to extract the total protein of cells in the culture solution:

1. Pour the culture solution into a 15 ml centrifuge tube and centrifuge at 2500rpm for 5 min.

2. Throw away the supernate, add 4 ml PBS and suck and spit for a few times with a pipettor to clean. Then centrifuge at 2500rpm for 5 min. Throw away the supernate and repeat the cleaning with PBS for once.

3. Remove the supernate with a pipettor and add 100 μl lysate (containing PMSF) to lyse on the ice for 30 min. Flick the tube often to lyse the cells thoroughly.

4. Mix the lysate with the lysate in the bottle and centrifuge under the condition 4℃、12000 rpm for 5 min. Supernate subpackage in 0.5 ml centrifuge tubes and preserve at -20℃.

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