Hello, ive been doing western blots and no bands show up, wece changed everythingdown to the transfer buffer, the blocking milk, antibodies and even the western blot machine. we still dont get bands. do you know what could possibly have this effect?
Hi Carolina, thank you for your message. There are many possible reasons for the absence of signals/bands (see below). For further trouble shooting, it would be great to contact me by e-mail (tech.service@serva.de). Looking forward to hear from you. BR, Judith • Inefficient Transfer o Check the transfer efficency by using pre-stained marker o Adjust transfer setting, e.g. duration if necessary o Use the appropriate transfer buffer, e.g. for even transfer of large and small proteins o Use the appropriate transfer membrane, do not forget to activate PVDF membranes with either methanol or ethanol • Antibodies may have lost reactivity o Check whether the antibodies are stored as recommended o If in doubt, use a new vial of each antibody • Non-fat milk may mask antigen o Test your Western Blot protocol by using another Blocking reagent, e.g. BSA, Casein or Protein-free like SERVA BlueBlock PF • Sodium azid contamination o In chemiluminescence detection the presence of NaN3 should be avoided! Check all solutions! • Contamination of stock solutions o Check the detection solutions for possible contaminations. o If in doubt, use new ones. • Incorrect Antibody o Check whether the primary antibody is the right one to detect your protein of interest and whether the secondary antibody recognizes the primary antibody • Low Antigen-Antibody Binding/Affinity o Make sure that the binding affinity of the antibody is sufficient for your assay. Further aspects: • Make sure that the substrate used for detection fits the enzyme label • Make sure that the detection system is suitable for your detection method
For this problem 6. Patchy spots E. Interaction with Sample Tray. What do you mean Sample Tray? Is it just the tray containing the membrane or the gel? Thanks in advance.
madxientist, you are absolutely right. To be concise, it is a matter of contamination or misbehaviour. Sometimes the plastic of a tray can interact with the AB due to contamination. The membrane can stick to the plastic surface and the solution can not swap around freely or the blocking reagent can interact with the plastic and causing sediments that stick to the membrane.
