Is it possible that my antigen will not elute in 2% SDS?

[markbond]

Hi Everyone,

have been trouble shooting an IP for my protein of interest for a while with not much luck. Using v5 tag and the Sigma anti V5 mono conjugated to agarose beads. I want to run Sypro Ruby stained gels for Mass spec ID of co-ip-ed porteins and want to avoid getting IgG heavy/light chains in the sample, hence the use of the conjugated Ab-resin. I have eluted in 200mM Glycine pH 2.5 at room temp, 3 times for 5 mins each - pooled and concentrated, followed by a subsequent incubation in 2% SDS, 10%glycerol, 50mM Tris pH6.8 at room temp. Did not heat this as I was afraid I would melt the agarose beads and release everything into the soluble phase. After running a sample on a western for V5 I had nothing in the elutions. however, the flow through had approx 70% less protein (i.e. the V5 tagged bait) than the total lysate sample, so my protein either stuck to the resin but did not elute or was simle degraded during the IP,even though I had protease inhibitors present.

My question: Is it possible that my protein will not elute from the anti V5 antibody in 2% SDS at room temperature? I thought this was pretty harsh but thinking about it I used to strip westerns in a similar SDS concentration but also with mercaptoethanol and heating and even then you could get some IgG still stuck to the blot?

Any thoughts?

Thanks

Mark
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[danfive]

Wash extensively and then try eluting with the SDS buffer--you've done low pH elution and seems it didn't work--SDS is foolproof, good thing the Ab is conjugated. I would elute in a low volume and keep my fractions separate--expect either the 1st or 2nd to be the most concentrated. Don't take any steps to concentrate or anything (keep it simple for now), try the Western straight from there, if you didn't carry over any beads then it should be fine to melt/boil protein before SDS-PAGE.

As to degradation PI works remarkably well (also depends on the properties of the sample of course). Just make sure the PI is relatively fresh and at reasonable concentration (1X according to manufacturer).

Pay attention to having fresh reagents--SDS, PI; and maximize the incubation period during antigen capture--if you believe to have proteases, 4-8 hours at 4C in presence of PI. If proteases are minimal then overnight at RT or 4C depending on what you know about your Ab.

[danfive]

Originally Posted by markbond

My question: Is it possible that my protein will not elute from the anti V5 antibody in 2% SDS at room temperature? I thought this was pretty harsh but thinking about it I used to strip westerns in a similar SDS concentration but also with mercaptoethanol and heating and even then you could get some IgG still stuck to the blot?

Any thoughts?

As long as the SDS is properly made (don't put it on ice), you will definitely separate the antigen-antibody complex. At least the great majority of it.

I've done a similar system of IP and MS for protein ID, and settled on the low pH Elution buffer because it was MS compatible. The SDS will need to be cleaned up. Another elution would be 3M Urea--compatible with WB, 2D gel, MS. No columns needed for buffer exchange/clean up.

[danfive]

Some tips from Pierce Product I used for IP

Antigen doesn't precipitate:
1. Cause: Sample doesn't contain enough antigen to detect (cell lysis incomplete, protein not expressed).
Solution Verify expression/lysis with SDS-PAGE or WB of crude lysate.

2. Cause: Antibody is sensitive to low pH and has lost activity (extremely rare).
Solution Elute with high salt, neutral pH buffer.

3. The antibody-antigen interaction does not elute using acidic conditions.
Solution Use neutral pH elution buffer, guanidine-HCl, urea, lithium bromide, potassium thiocyante or nonionic detergents to elute antigen.