Negative control for primary antibody in immunohistochemistry - What is it? (Sep/17/2008 )
It's become a big mystery to me how much of the fluorescence in my IHCs is specific. I had a control for the secondary antibody there is no problem with that. I know that the best control for the primary antibody is to use the exactly same tissue which does not express the protein though; unfortunately there is not such a tissue available in this case. The other way is to incubate the antibody with an excess amount of protein but this protein is too expensive (>$500) to be purchased only for one control slide. Does anybody know any other way?
This antibody can detect a band with the right size in western ...
a good negative control for your primary antibody is an isotype-matched antibody that won't bind in your tissue-of-interest. then you can be certain your binding is specific.
What is this isotop-matched antibody?
This is the second antibody I'm trying ( after a monoclonal antibody which couldn't even detect the protein in western) and unfortunately I'm facing some money and time restiricitons in this project; so I think my only option is to find some way to prove that the current antibody is working alright; I know it's not an easy thing to do.
This is the second antibody I'm trying ( after a monoclonal antibody which couldn't even detect the protein in western) and unfortunately I'm facing some money and time restiricitons in this project; so I think my only option is to find some way to prove that the current antibody is working alright; I know it's not an easy thing to do.
hala chera enghadr be in negative control ehtiaj dari?
I wouldn't bother thinking about negative control that much, don't waste your time on things that might delay your research. I'm not saying it's not important though, of course it is important, but you get my point, don't you?
some antibodies are not recommended for IHC or ICC. you need to check the company description for each antibody (which probably you are aware of it already). what protein are you trying to detect? there are tips to optimize your IF, ICC or IHC.
the best thing to do is to contact the person who has designed the antibody, when the antibody comes, a leaflet usually comes with it which has references to papers published with that specific clone of antibody.
movafagh bashid.
Che jaleb nemidounestam shoma ham irani hastin, khoshhalam ... !!!
I rather like to skip doing any control but my supervisory committee has asked for ... which I think that's fair; I am stating that this treatment does not have any effect on the protein expression in the tissue according to my IHC and they say "how do you know that your signal is just coming from your target protein? There might be lots of non-specific binding ( if not all ) which covers the specific signal ..."
If I had enough time and money in this project I would prefer to forget this antibody and try other antibodies from other companies ... right now though I have to stick with what I have in hand which is a polyclonal antibody not tested for IHC ( although cited once ), but at least can detect the protein from my weird animal (locust) in western ...
I have tried a whole variety of optimization steps which didn't really have any effect but, if nothing else, that's what I'm gonna back up my hypothesis with at the end ...
Thanks for your tips anyway ...
I rather like to skip doing any control but my supervisory committee has asked for ... which I think that's fair; I am stating that this treatment does not have any effect on the protein expression in the tissue according to my IHC and they say "how do you know that your signal is just coming from your target protein? There might be lots of non-specific binding ( if not all ) which covers the specific signal ..."
If I had enough time and money in this project I would prefer to forget this antibody and try other antibodies from other companies ... right now though I have to stick with what I have in hand which is a polyclonal antibody not tested for IHC ( although cited once ), but at least can detect the protein from my weird animal (locust) in western ...
I have tried a whole variety of optimization steps which didn't really have any effect but, if nothing else, that's what I'm gonna back up my hypothesis with at the end ...
Thanks for your tips anyway ...
what is the protein you want to detect? is it a secret?
Not at all, Hsp70 but in locust that's what makes all the differences ... 
geez ...
have you ever treated your samples only with your secondary antibody? I mean without your primary and only with the secondary. skip the primary treatment and only go for the secondary. do you think this would help?
If there is no negative control then that is not your problem, it is your supervisor's problem who is stingy and doesn't want to pay $500. but in case you observe the same fluorescence by only treating your sample with the secondary, then probably you don't have that protein in there at all.
Not that I know that this will work, but here's an idea: how about doing an immunodepletion experiment? You could make a GST tagged version of your protein, incubate the antibody with the tagged protein, then take the leftover antibody (whatever didn't bind to your tagged protein) and do your IHC. If the signal goes away, then you know that the original signal came from specific binding with the antibody. You would have to do a control (perhaps with a GST tagged version of some other protein) to show that the antibody wasn't degraded throughout the procedure. It may take a bit of time, but it wouldn't be too expensive to make - less than 500$. At any rate, you could propose it as an idea during your defense if they asked you for other ideas of how to prove your case.
Curtis, I've had such a control indeed; I eliminated the 1st antibody from one slide and everything else was exactly the same, the signal observed was very faint which shows that there is not any non-specific binding of secondary antibody ...
smu2, I like the idea of proposing some solutions in the defense ...