Suddenly my western blots are not working anymore. Protein transfer from SDS or native gels is good and many bands show up after Ponceau staining. In-gel activity assays of the native gels show activity.
Made all buffers new, got new ECL kits (Pierce ECL (#32209) and Pierce Supersignal (#37071)). I had to start new vials of primary and secondary antibodies, which all still have the same order number but different lot numbers.
The most signal I get is faint and quickly fades away. It doesn't matter, if I use less or more secondary antibody. Question: our secondary antibody is HRP conjugated and some of our primary antibodies have 0.02 % sodium azide added for preservation. If using a 1;500 to 1:1000 dilution and multiple wash steps I would expect that the concentration of sodium azide is very low to begin with and would be washed away. Any suggestions to solve my problem are appreciated.
Thank you
sometimes, if the signal is very strong, the positive band would fade away as the substrate is consumed locally.
Hello Gisela.
In those cases that have been working with no problem, and gradually not working, you could cast a question about the storage at 4C or -20C with frequent opening the fridge or freezer door and possible degradation of the all antibodies. Get new ones, aliquots in small eppen-tubes, store at -70C with 2mg BSA/ml PBS-diluted 2-fold as a carrier and preventing sticking to the walls of any container. Ignore the manufacture's instruction "Do not freeze". You won't fail any more.
Best wishes.
Hi,
It seems that you have already done a reasonable range of troubleshooting.
I agree with you that the azide in the primary antibody is unlikely to be the cause of the issue.
It''s not obvious if you have the problem with only one target protein, some of the proteins, or all the proteins you study.
How are other people in your lab? Do they have same issue?
These information may help to dissect where the problem could be.
Also, have you checked the detection method (film or camera)? Did you or somebody else notice a reduction in sensitivity?
Related to this, what is the detection method for the In-gel assay? Is it also luminescent? Does it share the instrument for the detection?
I think you schuld increse the target protein level in the gel. I think you have to make every time fresh washing puffer with good pH, The perfect pH (7,4) and the 1X fresh Buffer (25 mM Tris, 0,15 M NaCl, 0,1 % Tween 20) is very important. I think you schould decrease the wahing steps to 3X 5 minutes especially if you use R @ D antibodys, you can was the connected antibodies down.
If you are washing with TBST after the secondary antibody incubation just wash once with TBS (no Tween) after three times wash with TBST . Tween basically blocks HRP to show signal .
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