1. Western Blot Transfer Troubleshooting: No bands transferred to the membrane When none of the protein bands appear on the membrane, the most likely reason is problems relating to either the equipment or the assembly of the gel membrane sandwich. The polyvinylidene fluoride (PVDF) or nitrocel...
Overcome your Western blot difficulties with our troubleshooting advice, covering problems such as weak/no signal, non-specific bands, high signal, and other common issues.
Figure 1: Western blotting after IP – Anti-Light Chain specific antibodies to avoid obscuring analytes in the 50 kDa range. Gels were loaded with Mouse IgG, whole molecule (015-000-003) After SDS-PAGE and transfer to nitrocellulose, blots were blocked with BSA (10% w/v). After incubation...
Tackle high background staining on your western blot. Antibody concentration too high, Insufficient blocking, Inadequate washing and more.
I performed a western transfer and none of my proteins transferred to the membrane. Can you offer some tips? It is possible that the gel/membrane sandwich was assembled in the reverse direction such that the proteins ...
General western blot troubleshooting Nonspecific or diffuse bands Possible cause Solutions Antibody concentration too high Reduce concentrations of antibodies, particularly of primary antibody. Too much protein loaded on gel Reduce the amount of sample...
Protein transfer to the membrane has been inefficient Ensure the transfer was successful by performing staining of the membrane/and or gel. A reversible stain such as Ponceau S will allow you to blot your membrane after, or staining the leftover g...
Western Blot Troubleshooting Guide Common problems encountered in western blotting include low signal or high background. This video helps you troubleshoot problems in your Western Blot experiment, and provides solutions to ensure you get the expected results in the shortest amount of time. Read more...
附录一:Troubleshooting Blotting Problems(P43-48)附录二:Western Blotting Troubleshooting Logic Tree(P390-394)附录三:试验中所用到的试剂和缓冲溶液1 5x Stop Solution (Sample buffer pH6.7: Stock: to use: 20% Glycerol 100ml take 9.5ml stock 10% SDS 50g or 250ml 20% SDS add BME 0.5ml 250mM ...
Western Blot Troubleshooting Common as they are in today’s protein laboratories, Western blots are—let’s face it—a royal pain in the neck. For one thing, the method itself is lengthy and laborious: run the gel, transfer the gel, block, primary antibody, wash, secondary antibody, wash,...