Membrane proteins are protein molecules present in the cytoplasmic membrane of cells and play important roles in intracellular molecular transport, energy metabolism, maintenance of cellular and tissue structures, and cellular signal transduction. Currently, there are three main methods used for the extraction of cell membrane proteins: descaler and surfactant extraction, biotinylated membrane protein extraction, and the method of separating membrane proteins using glycans from cell membrane glycoproteins. There are three main methods used for the determination of the content of a membrane protein in cell membranes: immunoprecipitation, immunoblotting, and in situ flow cytometry using cells.
Principle
Biotin-label membrane protein isolation is a method of affinity purification of proteins, in which biotin is coupled to membrane proteins, and biotin-coupled proteins are isolated and purified by taking advantage of the high-affinity binding of biotin to affinities in egg whites and chain affinities in bacteria. Biotin is a water-soluble molecule that cannot pass through the lipid bilayer of the cell membrane, so the biotin molecule can only bind to cell membrane proteins. The 8-amino group on lysine residues in protein molecules, the sulfhydryl group of cysteine, and the aspartic acid and glutamic acid at the carboxyl terminus of protein molecules are the binding sites for biotin and its derivatives.
People have not been able to accurately quantify a particular membrane protein in cell membranes so far, and most of the methods currently used are based on immunological means, i.e., the principle of antigen-antibody binding, and relative quantification of cell-specific membrane proteins, in order to compare the changes in the content of the target proteins in the same cell after different treatments as well as the differences in the content of the same membrane proteins between different cells.
Operation method
Biotinylated membrane protein extraction method combined with immunoprecipitation for determination of cell membrane proteins
Materials and Instruments
Equipment: centrifuge, centrifuge tubes, protein transfer device Move The basic process of the biotinylated membrane protein extraction method combined with immunoprecipitation to determine the content of cell-secreted proteins can be divided into the following steps: A. Biotinylated membrane protein extraction method Discard the cell culture medium, rinse the cells once with phosphate buffer solution PBS (pH 7.4); add EZlink sulfo-NHS-SS-biotin working solution (PBS as solvent, freshly prepared) to the cells and incubate at 37℃ for 10 min; discard the reaction solution, add freshly prepared lysine solution and incubate at 37℃ for 5 min to neutralize excess biotin molecules; discard the lysine solution and wash with PBS. Discard the lysine solution, wash the cells with PBS once, and then lysed the cells with cell lysis solution containing 1% NP-40 to obtain the extract containing cell membrane proteins; purify the above protein extract by using affinityin or streptavidin agarose gel, and finally obtain the high-purity biotinylated cell membrane proteins. B. After obtaining the cell lysate, immunoprecipitation reaction can also be carried out with an antibody specific for the target protein to obtain the target protein with higher purity. 1. Divide the cell lysate containing the target protein into two equal portions and place them in two centrifuge tubes. Add an antibody against the target protein to one tube, and add a control antibody (either pre-immune serum or monoclonal antibody against a non-associated antigen) to the other tube. After mixing, put the two sample tubes in an ice bath and shake gently for 1h, or 2~3h if the supernatant of hybridoma is used as control. 2. Add sufficient amount of pre-treated Staphylococcus aureus A protein-sepharose resin to the above antigen-antibody mixture, mix well and place in an ice bath with gentle shaking for 1h. 3. Collect the S. aureus A protein-sepharose resin-antigen-antibody complex by centrifugation at 12,000r/min at 4℃ for 15～20s. 4. Remove the supernatant carefully, add 1ml of RIPA buffer containing 0.5mol/L NaCl to resuspend the precipitate, place it on a shaking table at 4℃ for 20min, and centrifuge it at 4℃ for 20s at 12 000r/min. 5. Repeat step 4 twice. 6. Carefully remove the supernatant, add 1ml of RIPA buffer containing 0.15mol/L NaCl to resuspend the precipitate, place on a shaker at 4℃ for 20min, centrifuge at 4℃ for 20s at 12,000r/min. 7. Carefully remove the supernatant, add 1 ml of buffer B to resuspend the precipitate, place it on a shaker at 4 ℃ for 20 min, and centrifuge at 4 ℃ for 20 s at 12 000 r／min. 8. Discard the supernatant and carefully blot the residual liquid on the precipitate and on the cap of the tube wall. 9. Add 1xSDS electrophoresis sample buffer, mix well, the sample is boiled at 100 ℃ for 5min, centrifuged at 12 000r/min for 5min, and the supernatant is transferred to a new centrifuge tube. 10. Take the obtained immunoprecipitate for immunoblotting analysis, using HRP-coupled streptavidin to incubate the transfer membrane, and obtain fluorescent bands by chemiluminescence to determine the position of the target protein according to the molecular weight of the target protein. For more product details, please visit Aladdin Scientific website.
Reagents:
①5xSDS-PAGE sample buffer
②
Electrotransfer buffer
③
③ Distilled water
④Deionized water
⑤ Buffer with SDS and mercaptoethanol
⑥1% NP-40 裂解缓冲液： 1 % NP-40, 50mmol/
L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1 mmol/L NaCl, 0.1 mmol/L NaCl, 0.1 mmol/L NaCl, 0.1 mmol/L NaCl, 0.1 mmol/L NaCl, 0.1 mmol/L NaCl.
0.1 mmol/L PMSF, l
mmol/L PMSF, lμmol/L pepstatin, 0.5 mg/ ml NP-40, 1 % NP-40
0.5 mg/ ml leupeptin , 0.3 μmol/L aprotinin .
⑦
Bromophenol blue indicator
⑧TBST (TBS (
Tris buffered saline
TBS (Tris buffered saline)
TBST (TBS (Tris buffered saline) /0.1 % Tween 20) solution)
⑨ECL reaction solution (equal volumes of liquid A and liquid B are mixed and used immediately)