I am using the LiCor Odyssey to visualize my blots. I am trying to decide which primary antibody is the best to use for future experiments. One primary is mouse and the other is rabbit, I have 2 different secondary antibodies and the LiCor lets you visualize two secondary antibodies at the same time. However, I am wondering if there is an issue using 2 primary antibodies that probe for the same protein?
I can't answer to your question but I have an advice for you. I hope it will help you.
When I want to compare different antibodies that recognize the same protein I run several times the same sample: 3ul, 5ul, 10 ul, and repeat this, don't forget to separates each group with a marker size (protein ladder); I do the transfer and after I cut the membrane (separate each group). Now, you will have exactly the same on each piece of membrane, the only parameter that will changed in your western will be the antibody primary and secondary. You will be able to compare the result (images you will received after the detection) and then to choose the best antibody for your work.
Good Luck
I can't answer to your question but I have an advice for you. I hope it will help you.
When I want to compare different antibodies that recognize the same protein I run several times the same sample: 3ul, 5ul, 10 ul, and repeat this, don't forget to separates each group with a marker size (protein ladder); I do the transfer and after I cut the membrane (separate each group). Now, you will have exactly the same on each piece of membrane, the only parameter that will changed in your western will be the antibody primary and secondary. You will be able to compare the result (images you will received after the detection) and then to choose the best antibody for your work.
Good Luck
There are a number of considerations here. First, both antibodies might be binding to the same epitope on your protein of interest and therefore competing against each other so you may not really deduce which is better. Second, if the two antibodies bind different epitopes, there may be a chance that there's not even space for both to adequately bind and lastly if your antibodies have some nonspecific binding, it might just be a mess. I think the way to go is just test each separately like Lauren mentioned.
Alexander is right. If you are testing multiple 1o antibodies against one protein in one blot, it will be a real mess. Sometimes It might work out of luck for one or two proteins. But I would not waste my time, effort and reagents. Moreover, it wont save your time and effort by combining two or more 1o antibodies for one blot. I would rather do what Laura has already described.
Hi, Brittany, I think it will be better if you use antibody for separate membranes.As have been mention previuosly, you may have a competition between 2 antibody. Just compare signal from mouse and rabbit primary antibody (with same protein amount and same sepoaration/transfer) and you will have a clear conclusion! Good luck!
Hi Brittany! Although there could be some steric hindrance between the two primary antibodies, I think that you can use them together just to have an idea of how they work. I used to do so to see simultaneously a phosphorylated protein and its total without performing an stripping. Nevertheless to quantify the bands, I would use only one of them to avoid any competition between antibodies. Good luck!
My view are also same like Laure D. Sultan and Alexander Braun.
It will be not better choice to blot for same protein by two different primary antibody on same membrane. It is better to do separately.
Good luck..
Check the epitope sequences and ensure that they don't overlap. If the mouse antibody is a monoclonal and the rabbit a polyclonal, you should not encounter a problem.
Also take into account the sample you are using. For example, if the antibodies were raised against the human sequence, you may see different results using mouse cell or tissue lysate vs human cell or tissue lysate.
Since mouse antibodies tend to give you a little bit more background with mouse lysates, visualize it at the 800 channel and the rabbit at 700. If you can, use the goat or donkey anti-mouse IRDye680RD antibody. This secondary tends to give you less background than the AF680 or IRDye680LT.
Good luck.
If you just want to avoid running multiple blots, cut them in half after transfer. That way you can test both primary and secondary antibodies in one run.
HI, yes I do agree with everybody above.
I had to deal with exactly the same issue.
One of the first things you always need to go for is to use an antibody raised to a different species to your sample origin as most times this would lead to increased non-specific background
Secondly, you need to check if the 2 antibodies you are testing bind in the same site in which case you will have competition issues which will give you false results.
Thirdly, it depends on the target protein and it's conformation if bound to something else in which case again it could possibly leads to another competition issue.
what i tend to do when i'm testing new antibodies in general, is load a couple of samples that i have protein to spare, in multiple wells on a gel, transfer and then cut the blot in multiples i.e. you will end up with 3 little identical blots if you ran a 10 well blot (include protein ladders too) then you can test you antibodies in parallel.
I hope that helps
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