![]() ![]() Without adequate SDS, the proteins are not negatively charged and will not travel through the cell.If necessary, run fewer gels and/or fewer lanes at once. Decrease the time between loading the first well of the gel and beginning electrophoresis.Sample has diffused away from the well prior to applying power Learn about methods for accurate total protein quantitation from cell lysates A: The gel was probed using Goat Anti-Mouse IgG (H+L) (115-035-003), revealing bands corresponding to both heavy chains (50 kDa) and light chains (25 kDa).Ensure that the protein concentration of each sample is accurate.Increase the acrylamide percentage of the gel.Ensure that the leads are in the correct orientation, as the electrophoresis leads to the power supply may be reversed, causing the gel to run upward.Decrease the amount of time the gel is run.Leftmost and/or Rightmost Bands of the Gel are Distorted.Protein Bands Too Close Together (Not Completely Resolved).Sample Doesn’t Sink to the Bottom of the Well Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose.But we still use gels, because electrophoresis remains an effective way to separate proteins - so that the results of antibody-based immunodetection can be fairly unequivocal.Ĭlick on the Sample Preparation and Gel Electrophoresis topics to read about the possible causes and remedies: Problems and Solutions Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem. Related Resources: Brochures | Application NotesĮlectric currents, wires, leads, combs, leaks… so many opportunities for trouble. In this section, you can find solutions to problems with blot background signal. Sample Preparation and Gel Electrophoresis The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. ![]()
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