2 replies to this topic

[rensky]

Hi guys!

I've been trying to coat a custom peptide onto Immulon plates because I am trying to develop an assay. Sometimes I get signal, sometimes I don't.

I'm wondering whether it will be because the coating of the "sample" is not done well?

I use 1x PBS + 0.02% Tween-20 for coating.

Any other coating buffers out there? How would I know which type of buffers are best for my peptide, sample, antibody, etc?

Thanks!

[jadefalcon]

Hi!

I used a carbonate-coatingbuffer:

0.1 M Na2CO3
0.1 M NaHCO3
pH=9.5

coated 100µl/ well of a 96 well plate (medium binding capacity, but maxisorp plates worked equally well) at 4°C over night.

worked for all kinds of proteins. if it doesn't work with your protein, may changing the pH wpoulod be a good idea.

mike

[rensky]

I was told that I shouldn't add blocking buffer when coating capturing antibody because the proteins in the blocking buffer will bind to the Immulon (coated) plates?

Does this mean that I should only block once in the protocol and just use phosphate buffer to dilute any antibodies I need to use in the protocol?

Thanks!