Can I use several primary anti-body before secondary anti-body?
Hi Trung!
I suggest you strip your membrane after each western blot. No reviewer will accept your approach. Using antibodies is problematic by itself because most of the time you get multiple signals that result not only from unspecific binding but also from artifacts and other parameters affecting your protein such as partial proteolysis, dimerization, intrinsic mobility alteration, posttranslational modification,…To this you have to add that optimal incubation conditions may change from an antibody to another. Other factors that I don´t have in mind right now.
Hi, Certainly you can use any number of antibodies and may even amplify the signal. However, the increased the probability of nonspecific cross-reactions.
Hi, You can use multiple antibodies together, but as Gavrilova suggested, its true that there is possibility of getting non-specific reactivity. I usually incubate with primary antibody one after another and then use secondary antibodies together (like anti-mouse and anti-rabbit etc against the primary antibodies.).
We generally not prefer to use several primay antibodies together.. but the thing is. it will increase non-specific binding. although U can try that...If u didnt get the proper result then better u use one and strip it and use other!!
Thank Natalia and Rakesh Dey. Your answer is exactly what I think about
Hi Trung!
I suggest you strip your membrane after each western blot. No reviewer will accept your approach. Using antibodies is problematic by itself because most of the time you get multiple signals that result not only from unspecific binding but also from artifacts and other parameters affecting your protein such as partial proteolysis, dimerization, intrinsic mobility alteration, posttranslational modification,…To this you have to add that optimal incubation conditions may change from an antibody to another. Other factors that I don´t have in mind right now.
There is doul antibodies already labeled with 2 different florescence labels like FITC and rhodamin, so you will not need to use the secondary antibody
Hi Trung,
as Ahmed suggested, whether you can use multiple antibodies will depend on your detection system. There are various protocols for labelling with multiple fluorescent labels.
I would not put multiple antibodies onto a western blot. You have a number of choices;
1 strip the blots after each antibody
2 cut your membrane up into regions containing the molecular range in which your target proteins are to be found, and do all antibody overlays on these mini blots.
3 add your antibodies sequentially if you know the relative strength of signal you expect. For example if you know one antibody gives a weak signal for a protein of 65kd and no signal around 20-40kd, and another antibody gives a really strong signal at 30kd, then put the 65kd antibody on, add the secondary, do the ECL, get the result, wash the membranes and add the 30kd antibody and so on.
We have been doing simultaneous multistaining Westerns in our lab..especially when we need to normalize using a housekeeping protein. All you have to do is to make sure to avoid inter-species cross reactivity of secondary antibodies. This can be done by applying a mixture of primary antibodies of mouse and rabbit origin, followed by a mixture of the secondary goat-anti mouse and goat-anti-rabbit antibodies. In my opinion, this should work for upto 2 Primary antibodies, however due to the limitation of species from which antibodies are produced no more than 2 proteins can be detected in one blot at a time. Also, you must make sure that the molecular weights of the proteins you are detecting are far apart, so that they can be identified as 2 separate bands on the gel picture.
Hope this helps.
Agreed with Abdelhalim Boukaba's suggestion. You should strip your membrane if you want to incubate another primary antibody.
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