Ⅰ. It mainly includes the following 4 basic steps：

1.Sample preparation.

2.Electrophoresis separation.

3.Membrane transfer of proteins.

4.IHC and coloration——protein detection.

Ⅱ. Solutions and reagents:

● 1×PBS.

● Modified RIPA buffer
Tris-HCl: 50 mM, pH 7.4 ; NP-40: 1% ;Na-deoxycholate: 0.25% ;NaCl: 150 mM ;EDTA: 1 mM ;PMSF: 1 mM ;Aprotinin, leupeptin, pepstatin: 1 microgram/ml each ;Na³VO⁴: 1 mM ;NaF: 1 mM

● 1×SDS sample buffer
62.5mM Tris-HCl (pH 6.8 于 25 ° C), 2%w/v SDS, 10% glycerol,50mM DTT,0.01%w/v bromphenol blue.

● transfer buffer
25mM Tris base, 0.2M glycine, 20% methanol(pH 8.3).

● 10×TBS
Prepare 1L 10X TBS: 24.2 g Tris base, 80 g NaCl;adjust pH to 7.6 with 1N HCl.

● Non-fat milk powder or BSA

● Methanol.

● TBS/T buffer solution
1X TBS, 0.1% Tween-20.

● Closed buffer（TBS/T） X TBS, 0.1% Tween-20 add 5% w/v non-fat milk powder or BSA.

● Dilution of primary antibody
1X TBS, 0.1% Tween-20 add 5% BSA ( PcAb ) or 5% non-fat milk (Mab).

● Pre dyed protein marker can be used to monitor the efficiency of membrane transfer.

Ⅲ. Sample preparation：

The original sample can be cell, tissue, culture supernatant, immunoprecipitate or affinity purified protein. The following is the treatment method of cell sample for qualitative detection of the target protein. Refer to the relevant literature for other sample preparation methods.

1.Culture cells or drug treatment.

2.Discard the medium, rinse the cells twice with 1×PBS, and remove the remaining medium.

3.Add 1×SDS sample buffer（6-well plate, 100 µ l /w or 75 cm 2 plate, 500-1000 µl/bottle ），scrape off cells and transfer to EP tube.（notice：Ice operation）

4.Ultrasound cuts DNA for 10 to 15 seconds to reduce the viscosity of the sample.

5.Boil the sample for 5 minutes.

6.Centrifugation 12000g, 5 min, supernatant.

7.Electrophoresis separation：
Upper sample 15 to 20 μl to SDS-PAGE (10 cm x 10 cm) electrophoresis.To quantitatively detect the expression level of a protein, Ripa lysate was used to lyse the cells. Collect the cracking liquid into the centrifuge tube and mix it on the oscillator for 4-15min，14000g centrifuged for 15min（4℃），abandoned sediment. The protein concentration in the supernatant was measured by Bradford method or other protein determination methods to adjust the volume and quantity of the sample. The internal or external reference was also needed for Western hybridization, usually beta actin.