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 Example: Smeared Bands Due to Protein Glycosylation: Western blot analysis of extracts from HDLM-2 cells, untreated (-) or treated with PNGase F (+), using CD83 (D8V7V) Rabbit mAb 99075 (upper) or -Actin (D6A8) Rabbit mAb 8457 (lower). If you see DNA migration issues or smearing after post-staining with GelRed or GelGreen, then the problem is not caused by the nucleic acid dye. Learn about methods for accurate total protein quantitation from cell lysates.Why is the non-specific binding detected after Western blot Protein ladder bands can sometimes. Ensure that the protein concentration of each sample is accurate. Why were there smeared plasmid bands on the agarose gel.Increase the acrylamide percentage of the gel. Solve your western blot problems with this western blot troubleshooting guide, covering high background, low or no signal, multiple bands, uneven staining.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.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: Related Resources: Brochures | Application NotesĮlectric currents, wires, leads, combs, leaks… so many opportunities for trouble. Sample Preparation and Gel Electrophoresis
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