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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Western blotting problem
Posted by: mightymouse (IP Hidden, New member, 1)
Date: August 2, 2005 04:31PM
I'm in desperate need of some help with a problem that I just can't figure out. I previously used a Santa Cruz antibody for my protein (65kd) and got a very clear, strong band. I recently started using a new antibody from Cell Signaling but got no band at all, and this is using the same samples as I used with the Santa Cruz antibody. The only change was the antibody. I don't think there's a problem with the transfer step since I do have an actin band on the same blot. I was wondering whether it would help to increase the antibody concentration. I would appreciate any other suggestions.
Re: Western blotting problem
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: August 2, 2005 05:46PM
mightymouse,
Here is something you may want to do: 1. increase the antibody concentration. 2. use a more sensitive detection system. 3. use a recombinant protein as your positive control to figure out the optimal condition for the detection limits of this antibody. 4. switch back to the old vendor or call tech support . Good luck.
Re: Western blotting problem
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 3, 2005 04:06PM
mightymouse, antibody quality can vary greatly from one supplier to another. we've had the same problem before. we switched suppliers once to try and save money. the 'cheap' antibody ended up costing as more, since we had to use it at a much higher concentration. if you have done everything in the experiment the same, it is likely that your new antibody is to blame. Increase the primary antibody concentration by half (if using 1:1000 try 1:500).
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