Incorrect secondary antibody used Confirm that the appropriate secondary antibody is used. In some cases, it may be necessary to increase the incubation time (e.g., incubate overnight at 4☌).Ĭ. Insufficient incubation time with the primary antibody A one-hour incubation at room temperature is usually sufficient for detection of most proteins. Avoid reusing primary antibodies whenever possible.ī. For weakly expressed proteins, it may be necessary to increase the concentration of antibody. Insufficient primary or secondary antibody being used Use the recommended antibody dilutions described on the product datasheet as a starting point for your experiment. Note that high molecular weight proteins may require a longer time to transfer.ģ) Antibody hybridization and wash procedureĪ. The conditions should be optimized according to the molecular weight of the target protein. Over-transfer Please adjust the electrical current and time frame for transfer. Reversible Ponceau S membrane staining is an easy step to confirm protein transfer.ī. Consult the instruction manual explaining usage of PVDF membrane before the experiment. PVDF membranes must be “activated” by exposure to methanol prior to transfer. Incomplete transfer Make sure the PVDF membrane remains wet during the transfer. Expression of the protein of interest is induced only under certain conditions Check the relevant literature to see if any treatment (e.g., starvation or chemical agents) is required to induce adequate protein expression.Ģ) Inadequate transfer of protein from gel to membraneĪ. The protein of interest is enriched in a specific organelle Biochemical fractionation of subcellular compartments may be necessary to detect this type of protein.Į. If the protein of interest is expressed in a tissue- or cell type- specific manner, be sure to choose this specific tissue or cell type for your experiments.ĭ. Low expression of protein of interest Increasing the amount of protein extract loaded on your gel may resolve this problem. Protein degradation Always add protease inhibitors to lysis buffer prior to cell lysis and perform protein extraction on ice to avoid protein degradation.Ĭ. Inadequate cell lysis Ensure that cell lysis and protein extraction are properly.ī. Our partner GeneTex supports researchers with a comprehensive selection of troubleshooting tips for many issues which can occur in a western blot (WB) experiment.ĭownload also the Western Blot Troubleshhoting Guide from GeneTex as pdf.īe sure to load proper positive and negative controls to ensure that the WB procedure is performed correctly.Ī.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |