Western Blot: specting the protein vs probing for 'house keeping'

[Guest christian]

Hi all,

 

For qualifying loading equal protein in a western blot... Is it absolutely necessary to also probe a 'house keeping' portein (i.e. GAPDH, actin etc) even though I spect all of my proteins?

[kixxer]

I´m sorry, but I didn´t understand your question there ?

Why do you want to put the housekeeping protein on the SDS-Gel? Just as positive control? Or are you trying to do some shift-assays?

 

greets,

kix

____________________

http://www.biologia.fi

[Guest christian]

Kix,

 

The housekeeping protein has resolved on the SDS-gel along with my protein of interest, tyrosine hydroxlyase. After probing for the protein of interest, one can re-blot for the housekeeping protein. When people do this, they express thier data (i.e., OD readings) as a ratio of protein of interest to the housekeeping protein. While others will not probe for a housekeeping at all and rely on just doing a spectrophotometric reading (by using Lowry method) of their samples as a positive control so that they have loaded proteins equally between wells.

 

My question is can I just spect my samples and not do the house keeping protein?

 

thanks,

 

christian