The initial high current will align small molecules quickly in the stacking gel to get a sharp band. Small peptides: High voltage 150 –300V, offers good resolution so long as the process is quick.Run the gel according to the following guidance:

We recommend adding frozen gel ice packs to the outer chamber, which will prevent the buffer from overheating and altering the pore size of the gel, which typically results in a protein 'smile' efffect at development. Do not overfill wells as this will lead to spillage into neighbouring wells and corrupt the results.Īdd the buffers to the relevant chambers and ensure that there is a good seal on the gel cassette chamber.

Note: On loading, take care not to poke through the bottom of the wells with the tip as this will distort the band at the development stage.

When both gels have set and the samples are ready to load use a pipette with a gel-loading tip to gently load equal amounts of between 20-40μg of each lysate per well.(Ensure to use a plastic comb to form the wells for lysate application.) Once the separating gel is set prepare the stacking gel.The following can be used as a guideline: Prepare the separating gels according to the protein size you are looking to identify. SDS-PAGE (Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis) Gently remove the tubes from the centrifuge and place on ice, aspirate and transfer the supernatant to a fresh microtube and keep on ice.Centrifuge for the lysate for 20 min at 12000 rpm at 4☌ in a microcentrifuge.Protein extract should not be so diluted that protein is lost or large volumes are needed to load onto the gel. Note: The volume of lysis buffer should be determined according to the amount of tissue being used. Ensure a minimum concentration of 0.1 mg/ml, and an optimal concentration ~1-5 mg/ml.Rinse the blade twice with another 2 x 300μl lysis buffer before agitating for 2 hours at 4☌ (e.g place on an orbital shaker in the fridge).For a ~5 mg piece of tissue, add ~300μl lysis buffer to the tube and homogenise with an electric homogeniser.The sample may be stored at -80☌ for later use, or keep on ice for immediate homogenisation.Place the tissue in a round-bottom microfuge tube or Eppendorf tube and immerse in liquid nitrogen to “snap freeze”.Dissect the tissue of interest with clean tools.Note: For this process we recommend keeping the tissue cool throughout the process to avoid degradation by proteases. Denature the lysate by heating the lysate at 95☌ for 5 minutes.Aspirate the supernatant and transfer to a fresh tube kept on ice, and discard the pellet.Gently remove the lysate from the centrifuge and place on ice.leukocytes need a very light centrifugation). For this step you should adjust the centrifugation force and time depending on the cell type - as a guideline use 20 minutes at 12,000 rpm, but this should be determined by the person carrying out the procedure (e.g. In a cool microcentrifuge, centrifuge the lysate.Maintain constant agitation for 30 minutes at 4☌.Scrape any adherent cells off the dish using a cold plastic cell scraper before gently transferring the cell suspension into a pre-cooled microfuge tube.(1 ml per 10 7 cells/100mm dish or 150cm 2 flask, 0.5ml per 5x10 6 cells/60mm dish or 75cm 2 flask) Remove the PBS and add ice-cold lysis buffer.Place the cell culture dish on ice and wash the cells with ice-cold Phosphate Buffer Saline (PBS).Check that the confluency of the cells is to expectation by viewing under a microscope.

Western blot protocol