I use TBS1X- Tween 0.1% for using primary antibodies for western blot. I had problems with one protein and a friend suggested me to try in 5% skimmed milk with sodium azide. The antibody worked well and also I got good bands. But the problem is after using that particular Ab in milk, I am not able to see any bands with other antibodies, even the loading control (which I use with TBST), inspite of stripping the blot before incubating with the Ab. What should I do? Should I use other antibodies also with milk?
Just a question: for blocking the membrane, what you use? 5% skimmed milk? and the membrane? is nitro or PVDF?
to answer your question..1-avoid sodium azide, it can interfere with the Ab binding.2-lower milk content (half, 2,5%) and see if it works.
Also keep in mind that some Ab (i.e vs phosphoprotein) does not work with milk, use instead BSA 5%.
Good Luck!!
Thank you.. I use milk to block the membrane and I use PVDF membrane
Ok, so, try lowering the milk to 2.5%. And another thing..which solution you use for stripping? Because some, mild stripping solution could not remove all (if you do not re-block after stripping it means it is too mild)
Hi, First, ensure that your primary antibody dose not make cross reaction with milk proteins. It's may forms Ab-milk protein junction. I recommend you try a experiment using 1% BSA in TBST as antibody diluent. Second, sodium azide inhibits HRP activity, If your secondary Ab is conjugated with HRP avoid sodium azide quite.
good luck
Thank you for your suggestions. I use commercial stripping buffer from thermo scientific and I reblock after stripping.
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