I used the antibody (against 14-3-3 theta) for immunoprecipitation (IP). When I use the same antibody to blot the sample from IP, it seems the light chain overlaps with the target band of 14-3-3. I came up with an idea of preparing the sample without the reducing agent (BME in my case) so that the light chain and heavy chain may stick together as a result of the intact disulfide bonds. I'm wondering if anyone have prepared samples for western blotting without adding reducing agent. What's your recipe of loading buffer (without reducing agent)? Do you boil your sample afterwards or for how long?
Another solution is to use secondary Abs recognizing only native and not denatured form of IgG, for example https://www.thermofisher.com/order/catalog/product/21232
Here is the recipe for a 5x protein sample buffer
50% (v/v) glycerol
0.25M Tris pH 6.8
5% SDS
0.05% bromophenol blue
In ddH2O
Mix it 1:5 with your IP sample, eventually adding a little H2O to make the right concentration
Cook it for a minute and it is ready for use.
Be sure to have the molweight marker and other lanes in the same buffer without reducing agents.
If you have the choice of another (species) Ab for the WB, you would do good to use that.
Good luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques