Include a molecular weight marker in one of the lanes. Load samples containing equal amounts of protein (10-50 μg/lane protein from cell lysate or 10-100 ng/lane purified protein) prepared in sample buffer into SDS-PAGE wells.Prepare or purchase a pre-made gel of appropriate polyacrylamide percentage to best resolve your protein of interest based on molecular weight.SDS-PAGE GEL ELECTROPHORESIS Protein size Note: This step should only be skipped if the antibody datasheet recommends non-reducing or non-denaturing conditions. Reduce and denature the samples by boiling the lysates in Laemmli Buffer at 95-100˚C for 5 minutes.Determine how much protein to load (Recommended: 10-50 μg/lane) and add an equal volume 2X Laemmli buffer.Determine the protein concentration of each cell lysate.Transfer the supernatant (lysate) to a fresh tube on ice.Note: The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. If required, the cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer. Using a cell scrapper, scrape adherent cells off the dish and transfer the cell suspension into a microcentrifuge tube.

See the table above for lysis buffer recommendations based on the subcellular location of the protein of interest.

Aspirate PBS and add ice-cold lysis buffer (1 mL per confluent 10 7 cells/100mm dish/150 cm 2 flask).

Wash cell culture dish on ice with ice-cold PBS.

Print a copy of our western blot protocolĬELL LYSATE PREPARATION FROM ADHERANT CELLS Next, secondary antibodies bound to enzymes are applied and finally a substrate that reacts with the secondary antibody-bound enzyme is added for detection of the antibody/protein complex.

The membrane can then be processed with primary antibodies specific for target proteins of interest. Next, the proteins are transferred from the gel to a membrane by application of an electrical current. First, proteins are separated from each other based on their size by SDS-PAGE gel electrophoresis. Because amino acid sequences vary from protein to protein, western blotting analysis can be used to identify and quantify a single protein in a lysate that contains thousands of different proteins. Antibodies bind to highly specific sequences of amino acids, known as epitopes. Western blotting uses antibodies to identify individual proteins within a cell or tissue lysate. Transferring protein from gel to membrane

Protein ladder western blot