Hello, I have been having a difficult time with western blot lately and I was wondering, what blocking buffer and antibody buffer should I use when using antibodies of Santa Cruz Biotechnologies? I want to use B--actin, but they don't provide a blocking buffer recommendation in the datasheet.
Blocking buffer is 5% milk or 5% BSA in TBST 1X?
It states that antibodies should be diluted in a ratio of 1:1000 but in what exactly? TBST 1X with BSA, TBST 1X with milk or just TBST 1X?
My washing buffer is Tris Buffered Saline Tween 20 (TBST 1X)
In the past I had success blocking a SC B-actin primary in 5% BSA in 1x TBST (.1% Tween). I would also recommend performing a titration the antibody (maybe try like 4 different dilutions with the recommended one somewhere in the middle) if you don't plan on re-using it at the recommended dilution.
The choice of blocking agent for western blotting depends on many simple factors and can be quickly made.
1. Skimmed Milk is a great option (usually 5%w/v). it is cheap. Easy to prepare.
It can be prepared in PBST or TBST.
In general, PBST can be used to prepare when the antibody is not detecting a phosphorylated protein but whenever an antibody is probing a phosphoprotein, one should use TBS or TBST.
Milk should be avoided as a blocking agent in cases where phosphoproteins are to be detected. Milk contains phosphorylated proteins such as Casein.
2. BSA is another option to block membranes. BSA (3%w/v) is usually good enough for blocking.
The antibody that is to be used should be diluted in 1% BSA in PBS or TBS to prevent background in the blots. This is done to ensure that all non specific antibodies that may bind to BSA should bind BSA before antibody is spread in the blot. In my hands, I have found blocking with milk gives cleaner blots.
Although blocking buffers must be optimized depending on the features of your protein of interest, 5% skimmed milk solution prepared in TBS-Tween (1%) is the most commonly used one for standard western blot protocols.
It also shouldn't be a problem if you tried to use 5% BSA-TBS-T solution for antibody staining or blocking your membrane. Maybe your western blot problems arise from sample collection step, but not about antibody dilution. If I were you, I would include a positive control to see whether antibody staining is the main issue. You can try different antibody dilutions to select best working one.
Good luck!
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