I'm not aware of any reagents like this for His-tags, but I'm going to follow this just in case there are some!

I have used a FlAsH tag for quantifying proteins in SDS-PAGE gels. But it requires a CCPGCC sequence on your protein, not a His-tag.

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Cellular-Imaging/High-Content-Screening/FlAsH-and-ReAsH.html

I've been searching quite a bit, and certain possibilities are as follows:

1.) NTA (nitriloacetic acid) linked to a fluorophore such as an ATTO dye (sold through Sigma)

==> However, this is not a covalent attachment

2.) Anti-His 1º antibody treatment, followed by chemical cross-linking

==> The entire antibody isn't likely to remain intact through SDS-PAGE, resulting in smearing of the shifted "band" above the unlabelled band

3.) Covalent labelling of a His8 tag with a reactive NiNTA fluorecent probe

==> Ref 1: Covalent fluorescence labeling of His-tagged proteins on the surface of living cells. Hintersteiner et al., 2008, ChemBioChem 9:1391-1395

==> Ref 2: Site-specific covalent labeling of His-tag fused proteins with a reactive Ni(II)-NTA probe. Uchinomiya et al., 2009, Chem. Commun. 39:5880-5882

==> Both of these require synthesis protocols.

I am open to any and all suggestions regarding modifications to the above, or anything that hasn't yet been mentioned.

If you want to do covalent labeling with a fluorophore distinct from GFP and then you want to run the products out on SDS-PAGE and then compare with densitometry, the CCPGCC tag mentioned above is your best bet. ReAsH is commercially available, it binds covalently with reasonable specificity and affinity, and it can clearly be distinguished relative to GFP.

We have used the Ni/NTA approach extensively in my lab for labeling His tags inserted into proteins expressed in cells, but the labeling is non-covalent and somewhat weak (Kd~ 100 nM for bis-NTA compounds binding to His10 tags) so it won't survive SDS-PAGE. At one time, I think BioRad sold a fluorescent staining solution you could use to post-stain SDS-PAGE gels to detect His-tagged proteins. However, in my hands, it didn't work well.

The last point is that if you are looking for mobility shifts with these sorts of reagents (including ReAsH), then the protein of interest will have to be rather small since these reagents themselves only 500 Da or so.

What is the question you are trying to answer that cannot be addressed with a standard Anti-His western blot?

I am working with an integral membrane protein with a GFP-His8 tag, reconstituted in proteoliposomes. I would like to determine the orientation of the protein in the membrane.

Membrane proteins are notoriously difficult to Western blot, which is why the GFP tag is useful, because you can observe in-gel GFP fluorescence (under standard denaturing conditions) to detect the presence and position of your protein.

The idea is to label the proteoliposomes (with something anti-His8) in the presence and absence of permeabilization, and observe a shift in the migration profile and/or different amounts of His8 fluorescence labelling. The position and intensity of the in-gel GFP fluorescence band will allow me to standardize values via densitometry as well as monitor potential shifting of the band.

It is for these reasons that something specific to the His8 tag would be preferred.

Ah I see. Knowing your specific problem is very helpful! Can you express your protein in cells and does it associate with the plasma membrane? If so, then the bis-NTA compounds we work with should provide your answer. We have found that bis-Cy3NTA labels control HEK-293T cells after they have been permeabilized, indicating that nonspecific (i.e. non-His-tagged) intracellular targets exist. However, addition of this compound to intact cells results in relatively little nonspecific labeling. If your protein is plasma-membrane associated and the His8 tag is located on the extracellular surface, then it should be labeleable with Cy3NTA and that labeling should be distinguishable from background. Lots of "ifs" I know but this type of experiment might provide you with some answers.

Yes, i was looking into NTA-fluorophore compounds. I did indeed come across the Cy-NTA compounds, but those all appear to require synthesis steps. Sigma sells NTA linked to various ATTO dyes. Unfortunately, they are all back-ordered until mid-December.

If you know of any sources or companies from which I could obtain Cy-NTA (preferrably Cy5-NTA, as its fluorescence characteristics are distinct from GFP), I would be extremely grateful!

The Cy3NTA compound we use routinely is clearly distinguishable from GFP and would probably be helpful to you. I would suggest you read our recent papers with the reagent and decide if you would like to try it for your experiments. If so, we can probably help you. If you are interested, we can continue this discussion offline.

Hi Salim

If I understand your problem correctly, you could hold down the tagged protein with IMAC. That is a tethered ligand holding a partially coordinated metal, allowing the open coordinate spaces to latch onto the His tags, and then immobilising the tagged protein while others keep moving.

If this concept interests you, you can let me know and I will provide some references.

Regards

Martin

Looking at the discussion of your problem now, indicates that immobilisation is not your answer.