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Coating Buffer for Sandwich ELISA - please help! (Jan/27/2005 )

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!

-rensky-

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

-jadefalcon-

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!

-rensky-

hi


if you work with peptide, you have to take special plates particularly if your peptide is smaller than 50aa. For example:
Immulon* Microtiter* 96-Well Plates and Strips, Thermo Electron but only
Immulon 4 HBX—Extra High Binding.
With immulon 2, i had a very weak or no signal. With immulon 4, very reproductive result.


The coating buffer (carbonate buffer) work well, overnight at 4C.
For blocking, i have used BSA or gelatine. there s no problem.
Avoid milk powder, In every case (ELISA ,WB) because of the reproductivity of your result.

-fuji-

QUOTE (jadefalcon @ Jan 28 2005, 08:51 PM)
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


Hi,

Does this mean that we can adjust the pH of this carbonate buffer to maybe pH 6?
I'm having problem as my positive control does not give much readings. I suspect my peptide was inefficiently coated on the plate.

Does anyone encounter similar problem?? Tried with PBS, doesnt work as well.

Thanks...

-cherrysnow-

I wouldn't change your pH because it may interfere with binding to the plate. I have always used pH above 9. Instead try adjusting the concentration of peptide in your coat. Also, the blocking step is very important.

-WAstate-

The two buffers I have used are carbonate, pH 9.6 and 70% EtOH, but this is only good for a very select number of antigens.

-swanny-

We've used mainly commericial kits and they've all used either a carbonate buffer pH9.5, a phosphate buffer (BD kits) at 4 degrees overnight or just PBS at room temp/4 degrees overnight (R&D kits) to coat capture antibody onto the Immunlon 4HBX plates.

Rensku, I wonder if you should leave out the tween from your coating buffer and wash the plate with PBS/Tween 20 (0.05%) before blocking.

Merry christmas,
Ceri

-Ceri-