Positive isotype control
Sofia Omari (Author)
Hello, I have problem with the isotype control. I'm trying to detect intracellular antigen in the leukocytes. I fixed, permed blocked the cells to get our antibodies into the cells. I used isotype control to distinguish positive and negative signal, however the isotype control gives always the same pattern as my sample. I tried several times with different procedures and it's the same. What do think the problem is?
mrxsf
Hi Sofia,
Could you be a bit more specific - what antigen are you staining for? I can see that you posted this some time ago so I imagine you have an answer by now but all the same! :-)
Steph
Sofia Omari (Author)
Hi Steph,
Thanks for your reply. I'm trying to detect TRPV1 antigen, the receptor of the hot chilli, inside the leukocytes. I have not get an answer yet, so I did not complete the experiment yet. Thanks again
Sofia
mrxsf
Hi,
Just a bit of background of what I do - I'm staining T lymphocytes for extra- and intracellular markers such as transcription factors and cytokines. I do it routinely and successfully (now!) but I've had a few things to overcome along the way. I'll suggest a few things that maybe you can try.
Firstly, are you sure you're getting adequate permeability? I started off trying self-made saponin/methanol/tween preparations etc and didn't get anywhere - I now use a BioLegend permeabilising buffer (it's actually marketed for Foxp3 staining but I use it whenever I'm doing intracellular staining and it's great). I guess it depends where your TRPV1 antigen is in the cell - if it's nuclear it might be worth investing in a commercial buffer, if it's cytoplasmic then saponin or something may well work fine. Try also running a control antibody to a nuclear or cytoplasmic antigen depending on where your antigen of interest is, to see if it's permeabilisation that's the problem.
Re: isotype binding - do you expect that TRPV1 is in all leukocytes (sorry - I really know nothing about this molecule)? Or is it only on a very tiny population such that you might not see an appreciable difference when overlaying histograms for example? If you compensate another channel and look on a two colour dot plot, can you see anything at all that is positive relative to isotype control?
Let me know if these suggestions are useful or if you think it might be something else. If you give me more details I might be able to suggest something else.
Kind regards,
Steph
Sofia Omari (Author)
Hi Steph,
TRPV1 receptor is placed across the cytoplasmic membrane. The primary antibody binding sites are either on the C- or N- terminus of the receptor (intracellularly). I tried different fix/permeabilise methods such as using tween 20 in PBS, Caltag TM invitrogen and Cytofix/CytopermTM BD Kits. I did blocking steps with BSA, human albumin and human AB blood serum and going no where. We did the compensation optimisation. We used secondary antibody control and it always gives negative results, however the isotype control for primary antibody was positive.
There is lackage in the literature regarding TRPV1 expression in leukocytes, so it may not be expressed strong enough to get a strong signal. Even though, isotype control should give negative result if the procedure is right. However, it did not. Here is our overlay histogram. Green colour is for primary+ secondary antibodies, pink colour is for isotype control with secondary antibody and blue colour is for secondary antibody control. I wish this will give an idea about my work. Thanks, Sofia
Sofia Omari (Author)
Hi Steph,
TRPV1 receptor is placed across the cytoplasmic membrane. The primary antibody binding sites are either on the C- or N- terminus of the receptor (intracellularly). I tried different fix/permeabilise methods such as using tween 20 in PBS, Caltag TM invitrogen and Cytofix/CytopermTM BD Kits. I did blocking steps with BSA, human albumin and human AB blood serum and going no where. We did the compensation optimisation. We used secondary antibody control and it always gives negative results, however the isotype control for primary antibody was positive.
There is lackage in the literature regarding TRPV1 expression in leukocytes, so it may not be expressed strong enough to get a strong signal. Even though, isotype control should give negative result if the procedure is right. However, it did not. Here is our overlay histogram. Green colour is for primary+ secondary antibodies, pink colour is for isotype control with secondary antibody and blue colour is for secondary antibody control. I wish this will give an idea about my work. Thanks, Sofia
AnisL
Hi there,
For such weakly expressed antigens, I would recommend you use the brightest secondary antibody you could. Using the same sample use as control a highly expressed antigen, such as CD45 which you will also stain with the same secondary antibody. This will tell you if it is a dye/antibody or other issue. Anis