I am using the Peprotech kit for IP-10 Elisa. I was using the standards provided by the kit and did serial dilutions, the last point of 0 pg/ml was used as diluent alone. However, when I was measuring my results I realized that some of the tested cells gave me lower O.D values than the O.D of the 0 pg/ml Standard. Thus when I calculated the IP-10 values in my experiment some of the tested wells had a negative value in terms of pg/ml. Has anyone experienced suce a thing? Is there any way to sove this issue? Also It took a while (about 30 min) until the color developed to give O.D values of 0.9 for the 1000 pg/ml standard.
I have found several protocols for adherent cell lysis buffer (NP-40). Some have 10mM,20mM or 50mM Tris HCl. Why is there a diference? Also, it was suggested that RIPA buffer is not compatible with Bio-Rad bradford assay reagent, however in the guidelines of the reagent it is mentioned that up to 0.1% SDS is compatible. Any recommendations for good lysis buffer protocols for both membrane and cytosolic protein extraction?