Hi Patrick,

According to Technical Bulletin of the Sigma product (#A2220) there are three ways of protein elution according to protein characteristics or further usage:

1. Protein elution under native conditions by competition with 3XFLAG peptide. The elution efficiency is very high using this method.

2.Elution under acidic conditions with 0.1 M glycine HCl, pH3.5. This is a fast and efficient elution method. Equilibration of the eluted protein with wash buffer may help preserve its activity.

3.Elution with sample buffer for gel electrophoresis and immunoblotting.

I usually elute by heating at 65°C overnight in the following Elution Buffer (50 mM Tris-HCl pH 7.6, 10 mM EDTA, 1%SDS) or by boiling for five minutes in protein sample buffer.

Have you already checked the troubleshooting guide and also the reagent compatibility for the M2 affinity beads usage?

Kind regards

Marcelo.

Hi Marcelo,

Thanks for the response. I am using native conditions competing with 3XFLAG. I need to protein for downstream use and it cannot be denatured (it is sensitive to low pH). I have been through the troubleshooting guide and all of my reagents are compatible with the M2 beads. My issue at the moment is that everything I've read says my protein should be eluted however no detectable protein is coming off the beads.

Thanks

It doesn't sound like you are doing much wrong.... Blocking the beads is a good idea (provided you've washed the excess away afterwards, leaving it bound to the non specific sites) and if you are sure that the Flag fusion protein is being expressed, the Flag tag is exposed and it is definitely there (stuck to the beads ie you've boiled the beads and run a western, which I think you have done) - you don't mention the conc of FLAG peptide? I appreciate you are following a protocol to the letter, but it can't hurt to take it up to a larger excess conc ie double what you are using, + prolonging the incubation with agitation. You mentioned salts in your buffer - you could increase the NaCl to 250uM in case you're getting some ion exchange effects. Good luck with it, but I must admit I haven't had this problem with Flag IPs.

I have always had success with the SIGMA FLAG kit. I elute with double conc. of 3x flag peptide, twice each for 10 mins. I do a final elution with glycine and rarely see any flag tagged proteins in this suggesting two elutions is sufficient. make sure you prepare  the beads well with the lysis/wash buffer (i use the EZveiw red beads for ease), you can add sodium thiocyanate to prevent non specific interactions. good luck!

Thanks for the input Richard and Johanna. In my last experiment I did double the concentration of 3xFLAG to 1.5 mg/ml and I eluted at 4 °C overnight. I'm going to start from the beginning and control for everything from binding to elution. This way I may be able to isolate my issue.

Thanks!

I am having the same problem with elution. Did you finally solve your problem, Patrick?

Thanks.

I wasn't able to get the FLAG elution to work with that protein. I ended up using a 6x-His tag with a magnetic bead spin column kit.

thanks. I had problem with magnetic beads. My colleague said she had elution problem with magnetic beads but solved the problem with anti-FLAG agarose beads.

Were you using the FL-peptide (F3290)? Its technical bulletin says that this peptide is not compatible for 3X-FL tagged fusion proteins. So my guess is that if your protein has 3X FL tag, this particular peptide would not work. They instead suggest to use 3XFL peptide (F4799). Hope this information helps.