circumsporozoite ELISA problem

[whizza]

I am trying to start up an ELISA to detect malariaparasites in the malaria mosquito (Protocol by Wirtz et al. 1987). I am trying to use the 0,5% boiled casein blocking buffer (so without using BSA). In the protocol that I have it simply states: suspend 2,5 g casein in 50 ml 0,1 M (0,1 N) NaOH, and bring to a boil. After casein has dissolved, slowly add PBS and allow to cool.

Problem is, the casein wont dissolve, I tried boiling it for over 2 hours, but still without succes. Has anyone done this ELISA? I would appreciate it if someone can help me solve this problem.

ELISA Sporozoite Detection.doc

ELISA sporozoite Solutions.doc

[gr8place]

I seem to remember that you can not boil casein, try warming it up to like 60C with stiring.

PBS/tween20 is often used as blocking buffer for ELISA.

[Immunologist]

I don't quite know about the dissolution of casein, but I don't see why it would not be dissolving even at room temperature... in a normal pH solution.

 

But on the other hand, you can try and use milk... often used in western blots, as one of its main protein is casein. So skimmed milk could also do.

 

I guess you try to avoid blood products, but if not, FBS 10% is also an excellent blocking agent.

 

PBS-Tween is NOT a blocking solution, it can only serve to wash. There is no protein in it and therefore cannot block non-specific interactions.

[chadn]

But on the other hand, you can try and use milk... often used in western blots, as one of its main protein is casein. So skimmed milk could also do.

 

I've alwaysed used dry skimmed milk, mixed in PBS for blocking.

[gr8place]

PBS/Tween 20 CAN be used, in many cases, for blocking in ELISA! I have used this for many BSA-sensitive ELISA assays, eg. TGF beta, IGF's, etc...Tween 20 acts as a blocker itself.