Protocol Online logo
Top : New Forum Archives (2009-): : ELISA and Immunoassay

Eliza and Antibody - (Apr/18/2012 )

Hi! I'm an undergraduate student who just started research and my pi wants me to do the following with the help of others and I'm not sure how:
- we need to coat Eliza plates (5 ug/well)
-we put 100 uL in each well
- we need 3 plates, 100 wells
-we have antibody- 1ug/mL
-HOW MUCH antibody we should take to prepare coating solution?

How would I proceed, how do you perform this process?

Thanks soo much!


Need to coat 5 ug/well / 0.1 ml/well stock should be 50 ug/ml (50 ug/ml X 0.1ml = 5 ug)

Plates are 96 wells x 3 plates x 0.1 ml need 28.8 ml of coating solution...but should make 35 ml (extra).

Your antibody concentration seems too dilute are you certain it is 1 ug/ml or 1 mg/ml? At 1 ug/ml you can not coat 5 ug/well with 100 ul. 100 ul of your stock ab solution would give you 0.1 ug/well. If it is really 1 mg/ml then it can be diluted.

35 ml (0.05 mg/ml)/ 1 mg/ml = 1.75 ml (of the 1 mg/ml stock) dilute to 35 ml (or add 33.25 ml of diluent)

Coat for 1-2 hrs RT or overnight 4C.

Then block (400 ul/well)...etc...


just realized it is 1 mg/ml of antibody haha sorry for the typo! Thankss sooooo much for your help, really appreciate it! :)