Question: Difference between Stopping reaction and measuring Abs max?

[Monaco]

Hello all,

I was hoping someone could solve the following:

I have an ELISA assay. Standard set up: Plate - Capture receptor - Protein (His tagged) - anti his HRP Ab

I use TMB as the substrate for the HRP. I measure the abs at 650 over 1hr.
I do not stop the reaction

The peaks reach a maximum and then decrease to 0 (formation of the 2 electron transfer diimine product).

I have been using concentration dilutions of the His-tagged protein and recording the MAXIMUM blue signal. These maxima occur at different times (it takes longer to get a maximum for lower concentrations),

My Question: Can anyone tell me if this is a problem? Should I be stopping my reaction at a certain time - I cannot see the advantage to stopping the reaction/ the disadvantage to not stopping it.

If it helps I am interested in observing a reduction in receptor - protein interaction

Any help would be great - Thanks!!

[Zagami]

Dear
An answer at your problem is explained in my post to which reference you
[Only registered users see links. ]

[butters]

Is this commercial kit? If yes, do follow the protocol set up by the manufacturer.

If not, I could be wrong here but not stopping it would not be wise. Stopping at the right time would be best.

Basically, the more His-Tag protein you have the more HRP ab anti HIS will bind to it. Thus allowing more rapid conversion. If less HRP ab present due to lower HIS-Taq protein it may still convert all/most the substrate at a much slower rate (longer duration).

I am slightly a bit confuse regarding observing reduction in receptor - protein interaction. you mean capture receptor - HIS-Tag protein?

Here you see is mostly HRP interaction with substrate unless you change your incubation time and etc etc... (i still can be wrong here).

[butters]

Zagami, your explanation is awesome! If I knew you are replying this, I won't need to think so long how to compose my reply!

[Monaco]

Hey guys,

Thanks for replying to me so quickly - I'm really stuck on this one. I think I almost have it figured out.

Butters - yes I am trying to measure a reduction between the interaction of the receptor and the his-tagged protein. At first I use my ELISA on the WT histagged protein, then I introduce a modification on the His-tagged protein and look for a reduction in the signal on my ELISA.

My problem is this: The TMB signal at 650 nm (which I currently measure) reaches a maximum and then reduces (formation of the yellow product). However, lower concentrations give lower absolute maximum (This max takes longer to get too, but it is still lower).

So, does this mean that I can relate the maximum at 650nm (independent of time) directly to the binding of the His-tagged protein?? Or should I be doing a kinetic ELISA and measuring the slope?

Cheers guys

[Zagami]

If your method is on Nickel Coated plate then :
Q. The TMB signal at 650 nm (which I currently measure) reaches a maximum and then reduces (formation of the yellow product)
A. Reaction go to saturation, then stopping is before time. In alternative can be presence of nonspecific binding of protein in target protein solution
Q. However, lower concentrations give lower absolute maximum (This max takes longer to get too, but it is still lower).
A. Add detergent to buffer containing the target protein to increase accessibility of the histidine tag.

[butters]

Yes everything seem clearer now!

[Zagami]

However, the binding between Ni-NTA and His-tag proteins is not very stable, and is often susceptible to interference by many commonly used chemicals and salts. Paborsky, L. R., Dunn, K. E., Gibbs, C. S., and Dougherty.: A nickel chelate microtiter plate assay for six histidine-containing proteins. Anal. Biochem. 234, 60–65, 1996