Thanks everyone ...
I am looking NOT to immunize with denatured protein, but rather to screen with
denatured protein via ELISA. IF I boil the protein we are concerned the
protein will fall out of solution, which would be fine for immunization but not
coating an ELISA plate.
Laura ....
With respect to Ni plates ... they are great ... but expensive (10x more than a
regular plate). We use them when we want the target to be as native as
possible, we have a HIS tag, and we do not have an antibody / tag combo for a
sandwich ELISA. I am not at all surprised the ELISA results are different.
Binding a well formed protein to plastic will alter it's structure. Had not
thought of using it as a screening protocol ... thanks.
On 5/25/20, 2:28 PM, "Antibody Discussion Forum on behalf of Bover,Laura"
<email obscured> on behalf of <email obscured>> wrote:
Hi Frances! Hope you and the group are well and hanging there!
Same than Ed. Denatured by boiling and use for injection and coat.
Sometimes I alternate injections: boil or not, in case I want to get the two
options.
One interesting alternative that I found for protein coated directly on the
plate (meaning "flat"?) is to use the his tag version of the protein and side
by side regular plates and Niquel coated plates (commercial).
The first time I asked my technician to do it, he was panicking because
although some of the wells showed identical OD, other showed very different and
some were negative/positive depending on the plate.
So, I use those plates for some projects, not as an alternative but
complementary.
Laura Bover
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Subject: [EXT] Re: [ABRF Ab Forum] Denature proteins
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Hi Frances -
The last few projects I have had to make antibodies to native (for FACS)
and denatured (for WB) protein I have found immunizing with the protein after
boiling it for about 1 minute has been effective for denaturing. The boiled
protein can be bound to ELISA plates or used in Dot-Blots.
Best,
Ed.
-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of
Frances Weis-Garcia
Sent: Friday, May 22, 2020 3:09 PM
To: ABRF Forum <email obscured>>
Subject: [ABRF Ab Forum] Denature proteins
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Yes. … it’s me again 😉
Looking for a protocol by which to screen denatured proteins. I have a
really nicely folded one and would like to see the antibody response against it
and a denatured version of it. Anyone have protocol they like.
I know an antibody core used to use a Urea protocol but they closed ☹.
Frances
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Yes. … it’s me again 😉 Looking for a protocol by which to screen denatured
proteins. I have a really nicely folded one and would like to see the antibody
re
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