amount of protein to load - (May/16/2005 )
Simple question but just wondering, how much protein do you all load per lane on a normal western blot? Normally I use 10ug per lane but recently not seeing much with my antibody. Wondering if I need to use more protein? Any ideas? what's the general consensus, is 10ug too little?
It actually depends on the protein that you want to detect. If you know that the protein is highly expressed then 15 ug is good. However, iI think the average that is loaded is around 20 -25 ug.
2nd: You may want to incubate the blot with the primary overnight or longer.
3rd: Make sure your protein has been actually transfered. High MW proteins might need a longer transfer time.
4th: I have also heard that if the milk used is old then the blot does not work.
5th: If it is a phosph-antibody, the phosphatase in the milk may be chewing it up. Try Bovine serum albumin instead of milk.
6th : If you are using the same antibody as earlier but a different stock then the company may be at fault.
Hope this helps!!
Thanks very much for that. I will try loading more protein and leave the antibody on for longer and see what happens. Thank you again
I generally load 10, 20 and 30ug of my protein for the first western to determine the amount to load on subsequent gels - however, i always find that 10ug is enough
It depends on your protein you're probing for. For one protein I only need to load 5ug to see a huge band but for the other protein, I have to load 40ug to even see it.