loading protein ladder for western blot transfer

Place the tissue in round-bottom microcentrifuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze.Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.

You may have to vary the centrifugation force and time depending on the cell type a guideline is 5 min at 14,000–17,000 g but this must be determined for your experiment (leukocytes need very light centrifugation). Centrifuge the cell suspension in a microcentrifuge at 4☌.Maintain constant agitation for 30 min at 4☌.Alternatively, cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer in a microcentrifuge tube. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube.Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 10 7 cells/100 mm dish/150 cm 2 flask 0.5 mL per 5x10 6 cells/60 mm dish/75 cm 2 flask).Place the cell culture dish on ice and wash the cells with ice-cold PBS.View our western blot protocol video below. Solutions and reagents: running, transfer, and blocking buffers.The membrane can then be further processed with antibodies specific for the target of interest and visualized using secondary antibodies and detection reagents. The gel is placed next to the membrane and the application of an electrical current induces the proteins to migrate from the gel to the membrane. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). Western blotting is a technique that uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. Our western blot protocol includes solutions and reagents, procedures, and useful links to guide you through your experiment.

Loading protein ladder for western blot transfer