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What is the best antibody for the housekeeping protein (control) in skeletal mus - (Nov/18/2014 )

Hi everyone

 

I am doing western blot for skeletal muscles samples. I tried different antibodies (beta-actin, alpha-actin & alpha-tubulin) for housekeeping protein (HKP) (control).

The problem is some of the samples do not express the HKP but my target proteins were expressed in all my samples. Example, out of 8 samples, only 4-5 samples expressed the HKP.

Please can anyone help and explain what could have caused this problem. I will also appreciate suggestions on the antibody I will use. Thanks

-Ubala-

Muscle tissue has high levels of actin - you should be able to detect it in all muscle samples.

 

If you can tell us what you did (details are the key to this), that would help us troubleshoot.

-bob1-

Sample preparation

 

The total protein loaded was 20ug at the first time and reduced to 15ug. The amount was calculated based on the concentration of each samples.

Exact amount was eliquoted and 1x PBS was used to equalized the volume of all samples to 10ul.

Then I used 2x Laemmil buffer (10ul) and the total volume of the protein (with the buffer) was 20ug.

I heated the samples at 90oC for 10mins. Then, samples were allowed to cool down for 5-10 minutes, at room temperature before loading in to wells.

I used 12% for resolving gel and 4% for the stacking gel. 

-Ubala-

Ok - mostly fine. Try diluting each sample in lysis buffer rather than PBS, this should balance the salt a little better.

Boiling samples for 10 min can cause them to precipitate (most abundant will come out first...) try 5 min, or try 70 C for 20 min.

Cool the samples on ice immediately following boiling and then spin briefly to remove any precipitate.

-bob1-