loading controls for western. which one to choose? - (Mar/26/2007 )
Hello, I am now jumping into a quantitative western blot business thus loading control becomes extremely important for my result. I've tried beta-actin but it seems not working well. Beta-tubulin may be a good choice, however I am not sure about how to choose the right one for one particular cell type.
Does anybody have experience on looking for a good loading control? any thoughts? I've been googling for a while but...
I appreciate very much for your help!
Annie
hello there
cycle,among different cell types and tissues at all stages of development, and unaffected by experimental treatments.
Thus, housekeeping gene–coded proteins are often chosen to serve this purpose. However, a number of studies
on gene expression showed that the expression levels of many housekeeping genes, such as beta-actin and GAPDH,tubulin can be
varied according to the cell type and the expierement you are doing.
hope this help .
Hi meflower,
I think you can try to use a molecular chaperone as a loaing control (such as DnaK or GroeL for bacteria). At a normal conditions (no shock, e.g. heat shock), the expression level of molecular chaperone is always constant.
Good luck!
Nam
Exactly! House-keeping gene expression may be different! That's the point! Thank you very much for pointing this out!
I guess that the only way for me to find one constant-expressed gene is trying different things until CV is small enough.
cycle,among different cell types and tissues at all stages of development, and unaffected by experimental treatments.
Thus, housekeeping gene–coded proteins are often chosen to serve this purpose. However, a number of studies
on gene expression showed that the expression levels of many housekeeping genes, such as beta-actin and GAPDH,tubulin can be
varied according to the cell type and the expierement you are doing.
hope this help .
Thank you for your help, Nam! Do you have any experience with anti-DnaK antibody? Which company did you buy from? I've had a lot of problems with santa cruz antibodies and would like to find out a good Ab before spending $$$$ on it.
I appreciate very much for any detailed info.
I think you can try to use a molecular chaperone as a loaing control (such as DnaK or GroeL for bacteria). At a normal conditions (no shock, e.g. heat shock), the expression level of molecular chaperone is always constant.
Good luck!
Nam
Hi meflower,
I often purchases my antibodies from AbCam. You could visit its website for Hsp70 antibody (DnaK antibody):
http://www.abcam.com/index.html?pageconfig=searchresults
And you also could find antibodies specific for other molecular chaperones at there
Good luck
Nam