Hi,

Firstly, what cells are you using - just checking it is not a mouse cell line as then your secondary is likely to label it up.

Secondly, what are your concentrations for the primary and secondary antibodies? Have you tried labelling with various dilution factors of the antibodies? In addition, have you just tried adding either primary only or secondary only and then viewing under microscope? Sometimes you get high background because of non-specific labelling due to high concentrations of the antibodies being used. Normally I tend to use 1:100-1:300 for primary and 1:200-1:500 for secondaries. Varies depending on quality of antibody.

Thirdly, what is your antibody diluent comprised of? Are you using something to block non-specific binding? I use a bit of normal goat serum in my diluent (as long as not using a goat antibody).

Finally, I'm guessing you've transfected your cells with nucleic acid that will express your his-tagged protein? Any chance you've cross contaminated your negative control cells?

Hope it helps!

Resonating with John, while you may be observing some non-specific binding due to inadequate titration of your antibody, you're description of "dense & similar" patterns of staining does sound as if your negative controls are contaminated. 

Some variability in staining can be observed between different his-tagged proteins, and may be contributing. But, I'd start with verifying your control wells are indeed without his-tag contamination, and titrating your antibody with at least a 5-pt or preferably 7 or 9-pt dilution series.

Hi

Thanks you John and Joshua for your reply.

I am using mammalian cell line (HUVEC). I am using the primary and secondary antibody dilution of 1:500. The diluent i am using is 0.25 % BSA / PBST and for blocking step i am using 1 % BSA/PBST.   In my experiment i am adding exogenous his tag protein  and analyzing for it internalization into the cell.

I will try doing the antibody titration and see any difference is come up. 

Thank you 

Hi

Did you manage to answer this question? I have been through numerous anti-his antibodies, all of which have shown non-specific nuclear staining. I have tried all the standard protocol alterations (altered blocking, fixation, cell-lines). I am now changing to an antibody raised in goat (instead of mouse) to see if this can alleviate the issue.

All appropriate controls have also been included in my experiments.

Hi

Many DNA binding proteins have leucine zipper etc motif that have positively charged motif - like a stretch of 6His residue. His Ab's may recognize DNA binding proteins and stain in the DNA. A great idea can be addition of 10-25 mM Imidazole in the buffer system while staining etc. This is greatly used in protein purification. Better blocking will help too.

Hi David Peeney,

As you stated i tried multiple ways to solve this issue. In my experiences most of the human cell lines gives out non-specific binding to His-tag antibody. Literature says that nuclear compartment contain proteins with long His residues (3 - 11) in stretch, hence trying Imidazole might not help to overcome the issue. With my limited knowledge i will suggest, not to plan IF experiment using His tag primary antibody against human cell lines.

Following this post....

I have encountered a similar issue with anti-6x-His antibody recently; even controls (no target protein present) show positive signal. The cells (A549) were treated and fixed in 4% PFA but not permeabilized as my target is a membrane-bound protein. Blocking was with 5% BSA for 1hr at RT; primary ab (anti-HIS(6x)), made up in blocking buffer, incubation overnight; secondary ab in blocking buffer-incubation at RT for 1 hr.

I also made a weird observation in this staining; not all cells show up positive nuclear staining (both in control and treatment), about 50% cells don't show up nuclear staining. I have attached a couple of images here that shows both DAPI and secondary ab channels.

Any comments/suggestions would be greatly appreciated.

Thanks.

Hi Thiru Sabapathy, thanks for following the post.

My suggestion is don't plan immunofluorescent staining using HIS tag antibody (Reason you can see in my 2019 jan reply). Even if you search pubmed you will hardly find good articles with HIS tag IF images. So smartly change your plan of action and use tags like Myc, flag for staining. I have lost nearly 4 months in optimizing, so don't do that again.

In regards to your staining, based on the images you shared... in both images, cells in centre of the field are bright and cells in left side seems to be dull.

This is most probably due to issue with your microscope stage, so check your stage is not wiggling and if you used oil immersion objective piece, oil is applied evenly... Hope my suggestion help you..

Thanks Renganathan !