Hi Jaya,

this is probably going to sound like a stupid question, but do you know definitively that you have successfully fused your protein to GFP?  Are you able to see that your cells fluoresce when illuminated with the appropriate light-source, and is your protein detectable on a Western blot with anti GFP antibody at the appropriate molecular weight for the fusion?

My understanding from collaborators who have used anti-GFP nanobodies is that they do work well generally, so if your protein isn't binding, that suggests something is going awry, whether during the IP or in terms of conformation of the GFP moiety.  On this latter question, does un-fused GFP control bind the beads in the absence of your protein? 

good luck, and best wishes,

Robin

Yes. The western looked good. I was thinking that 1 he might have been less time for binding the proteins. Could u shed any light on this

Sorry - I don't understand the question as written above - I assume that autocorrect has mangled the English.  

Can you rephrase it in context of the protocol that you had to follow?  My understanding from colleagues here is that when used for IP of cell lysates or bacterial extracts with GFP fusions, the binding / mixture time doesn't have to be particularly prolonged as the affinity of the nanobody for the GFP fusion is pretty high under most conditions.

best wishes,  Robin

Sorry. It was auto correction by the mobile. When I did the IP last time for one hr incubation the binding was not that much. Most of the protein was in the flow through, in fact the amount of protein in the flow through and the lysate before IP was approximately the same. That is why I was thinking to increase the incubation time overnight

Hi Jaya,  autocorrect happens!  :)

What I will say straight away is that if you have not encountered issues with visible degradation on the analysis gel (in the flowthrough), there should be no harm in increasing the binding time overnight in the cold room.  There were many occasions when I was a PhD student performing IPs when the plan to perform a 3 hour IP became overnight, or an overnight IP became an over-weekend IP and the experiment worked perfectly well.  I should stress though that this was using anti-myc or anti-HA antibodies, not the Chromotek one.

A GFP-only control, or positive control using another GFP fusion to account for possible steric effects between your protein of interest and the GFP moiety as regards its binding to the antibody-resin would be an important place to start if you have any doubts about the antibody / IP step.

best of luck, Robin

Thank you for the reply. I did an overnight incubation, but there was a lot of degredation on the western. I'm suspecting that the bound protein did not dissociate from the beads even after heating for 10 min at 95 deg. I'm planning to use glycine elution. Hopefully this works

Hi Jaya,

Did it work with Glycine elution?

Best regards, Raj

Yes. But not good as much as sds elution. My mistake was that I heated the beads at 95 deg and since mine was a membrane protein I had no product, but got it wen heated it for 65 deg for 20 min. 

Hi Jaya,

I was looking to get the GFP-trap from Chromotek also. Does it work?

Yes. It does. Perfectly with my protein

Great!,

Thank you Jaya!

I didn't understand. Why did elution at 65 degrees for 20minutes worked better? I'm also working with membrane protein, and I'm trying to optimize my IP.

That is because membrane proteins form aggregates at high temp, in this case 95 or 100 deg used for Western and will get stuck in the stacking gel

Hello Jaya,

May I kindly ask what's the consequence of the aggregation of membrane protein?

I am working on a membrane protein, and I always used 95 or 100 deg to denature it. Now I could get it in the western blotting, but I noticed that my protein ladder would be thinner a bit by being squeezed by beside loaded protein of my interest. I am not whether this is caused by the aggregation of membrane protein.

Thanks a lot!

This definitely is due to the aggregation of proteins. Depending on the experiment, I either heat it at 60deg for 20 min or after adding the lamelli buffer, I leave at room temperature for 20 min. There is a lot of difference between both. If ur ptn is a multimer u might want to see the difference both the condition. If u want to see weather the ptns are aggregated after heating to 95 is to stain the stacking gel. If it's aggregated u will see in the stacking gel. Just make sure to cut is a little bit with the running gel so as to see the results clearly

Jaya Aseervatham when you boiled at 95 degrees could you still separate your beads magnetically? It looks like mine melt or form a homogenous solution

Hi, I also tried to elute my protein using the magnetic gfp trap beads. While the protein band in SDS-PAGE appeared for the input fraction (cell lysate) as well as the flow through, no binding with the beads was detected. Can someone please suggest how to troubleshoot this?

Thanks in advance!

ChromoTek has a very good technical support team. Just send an email to [email protected]

Divya Dhasmana can you see your GFP-labelled protein? The GFP antibodies are normally exceptionally effective - to the point where you can watch affinity resin pulling GFP-tagged proteins to the side of a tube out of solution in real time with a blue light box - presumably magnetic beads you could watch the protein moving to a magnet outwith the tube if binding is taking place normally. I use visual realisation of GFP-labelled proteins during purification as a routine method to get instant feedback on conditions when working with new proteins in particular.

Robin Dickinson Thank you for the suggestion. Yes I could see the gfp labelled protein expressing in cells under fluorescnece microscope. On the SDS gel the protein was detected in the lysate (input fraction) and the flow through- as the prtoein band correponded to the protein mol weight. Can it be possible that the protein bound to the beads was too less to be detetced in an SDS PAGE? But I think that the binding did not place at all as the protein was present in the flowthorugh.

Christoph Eckert Thank you for the suggestion