![]() ![]() Acrylamide stock solution: 29% acrylamide plus 1.0% bis-acrylamide.Separating gel buffer: 1.5M Tris-HCl, pH8.8, 0.4% SDS.Stacking gel buffer: 0.5M Tris-HCl, pH6.8, 0.4% SDS.The cell lysate is evaluated as qualified, if the WB image shows five bands. The primary antibody we used is the antibody against the marker proteins. The cell lysate is evaluated as qualified, if the bands are clear and have no obvious smear. Incubate the lysates at 100☌ for 10min.Ī) Test cell lysate by SDS-PAGE. b) Mix the cell lysate with SDS loading buffer to make the desired final concentration.Measure protein concentration using cell lysis-compatible protein assay (BCA protein assay). Dilute the supernatant lysate and the whole lysate without centrifugation at 1:4, 1:8 and 1:16 with 1XPBS. Then collect supernatant into an appropriately labeled tube (for the detection of membrane bound proteins, use the insoluble, cell pellet). Keep the entire operation on ice.Ī) Transfer contents to a microcentrifuge tube. b) Disrupt tissue with ultrasonicator.Vortex the mixture for 5min and store at -20C.Ī) Thaw the frozen, raw tissue sample by vortexing and repeat the freeze-thaw cycle twice. Ĭ) Add cell lysis buffer to cell extracts (1ml lysis buffer /5×10 7 cells). When the concentration reach 10 6 cells/ml, detach cells, spin down and wash twice with 1XPBS. ī) Incubate cells for about three days and count the cells. Then spin down cells and re-suspend the pellet in DMEM with 10% FBS to make the final concentration around 105 cells/ml, and incubate at 37C, 5%CO2. Ii) Adherent cells: digest adherent cells in Cell Detaching Trypsin Buffer. I) Suspension cells: spin down cells and re-suspend pellet in RPMI 1640 with 10% FBS to make the final concentration around 105 cells/ml, and incubate at 37C, 5%CO2. Cell Lysis Buffer (1% NP40, 0.5% DOC, 1mM EDTA, 65mM Tris-HCl pH=6.4, 1mM PMSF, 1ug/ml Aprotinin, 1ug/ml Leupeptin, 1ug/ml Pepstatin).Cell Detaching Trypsin Buffer: 0.25% trypsin in 0.53M EDTA solution.SDS-Loading Buffer: 0.05M Tris-HCl, pH6.8, 0.1M DTT, 2%SDS, 10% glycerol, 0.1% bromophenol blue.Phosphate Buffered Saline 1X PBS, pH 7.4.Please consult the product data sheet for the appropriate concentration of primary antibody and any other special conditions. see example western blots from our validation program! Procedure: Enzyme substrate is then applied and the membrane is visualized for the presence of signal. The membrane is then probed with an antigen specific antibody which is then itself detected using an enzyme conjugated antibody. Typically, protein samples are resolved by their size by gel electrophoresis and transferred onto a membrane. Western Blotting / Immunoblotting (WB / IB) Description:Ī western blot is a technique used to identify the presence of an antigen in a particular tissue homogenate or protein extract. Western Blotting/Immunoblotting (WB/IB) ProtocolĮnzyme-Linked ImmunoSorbent Assay (ELISA) Protocolīlocking Peptide Competition Protocol (BPCP) High Throughput Antibody Characterization Services.Loading Control Antibodies for Western Blot.Protein on Demand™ Semi-Custom Recombinant Proteins. ![]()
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