amount of protein to load for a western? - (Sep/16/2008 )
hello all,
ive seen huge ranges from 3-30 ug of protein needed for western blots. anyone have some insight/advice? my wells have a 30 ul capacity and the sample with the lowest protein concentration i have is 0.275 ug/ul - im worried i wont be able to load an appropriate amount of protein if i need near the upper range that i mentioned.
any advice?
-medstudent1-
It depends on how abundant the protein of interest is present in samples. If your protein is expressed a lot, then loading small amounts is sufficient else more sample might be needed or a better antibody might help.
-scolix-
QUOTE (medstudent1 @ Sep 16 2008, 09:51 AM)
hello all,
ive seen huge ranges from 3-30 ug of protein needed for western blots. anyone have some insight/advice? my wells have a 30 ul capacity and the sample with the lowest protein concentration i have is 0.275 ug/ul - im worried i wont be able to load an appropriate amount of protein if i need near the upper range that i mentioned.
any advice?
ive seen huge ranges from 3-30 ug of protein needed for western blots. anyone have some insight/advice? my wells have a 30 ul capacity and the sample with the lowest protein concentration i have is 0.275 ug/ul - im worried i wont be able to load an appropriate amount of protein if i need near the upper range that i mentioned.
any advice?
it depends on the relative amount of the antigen and the quality of the antibody; we commonly use 15 µg per lane in mini gels; however, if you have good antigen expression and a top antibody, you can use smaller amounts; in such cases, we also succeeded with 8 µg per slot
-The Bearer-