Shanghai Biochip Co., Ltd.
Shanghai Biochip Co., Ltd.
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The test object is a tissue sample. Take an appropriate amount (about 150mg) of a fresh tissue sample or a properly preserved tissue sample, add 0.3-0.4ml total protein extraction reagent (or nucleoprotein extraction reagent) containing a protease inhibitor, and homogenize the ice. Extract the total protein (or nucleoprotein) by lysing and centrifugation.
The experimental object is a cell sample. Take 1 × 10 6 ～ 1 × 10 7 cells per sample, wash the cells with PBS to remove serum and other culture additives, and remove 0.1-0.2ml total protein extraction reagent containing protease inhibitor in PBS ( Or nucleoprotein extraction reagent), lysed on ice, and centrifuged to extract the total protein (or nucleoprotein).
Follow the instructions of the BCA Protein Quantification Kit to determine the sample concentration.
3. Denatured polyacrylamide gel electrophoresis (SDS-PAGE)
Load the prepared sample solution and the pre-stained protein marker separately, add the standard to the first well, 80v electrophoresis concentrated gel, 100v electrophoresis separation gel.
4. Protein transfer to NC membrane
Assemble the filter paper gel cellulose membrane sandwich (Vinyl white film sandwich model) according to the instructions of the Bio-Rad protein transfer device, and transfer the membrane under the constant pressure of 100v, and select the membrane transfer time (1KD / 1min).
5. Western blot membrane blocking and antibody incubation
The membrane was incubated in a 5% skimmed milk powder solution for 1 hour at room temperature to block non-specific binding on the membrane.
Add the primary antibody (including GAPDH or Actin internal reference antibody) to the blocked membrane and incubate at room temperature for 1.5 hours. The antigen and antibody will bind. Wash three times with 0.15% PBST for 15 min each. Incubate the fluorescent secondary antibody for 1 hour at room temperature in the dark.
Wash three times with 0.15% PBST for 15 min each.
6. Western blot results
For fluorescence detection, the fluorescence intensity of protein bands was detected using a LI-COR ODYSSEY protein imager.
7. Western blot data analysis
The gray value of the target protein is divided by the gray value of the internal reference GAPDH / Actin to correct the error. The result represents the relative content of the target protein in a sample.
8. Provide experimental report
Includes detailed experimental methods and relevant data for western blot results.
Immunoblot, also known as Western blot, is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. This method is a new immunobiochemical technology developed on the basis of gel electrophoresis and solid-phase immunoassay technology. Due to the high resolution of SDS-PAGE and the high specificity and sensitivity of solid-phase immunoassays, immunoblotting has become a routine technique for protein analysis. Immunoblotting is commonly used to identify a protein, and enables qualitative and semi-quantitative analysis of the protein.
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