Western blot - choosing correct antibody
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Western blot - choosing correct antibody
Hello, I need a help with my western blot experiment. Although I read a lot western blot guidelines online, I still can´t decide how to design my experiment, especially how to correctly choose my antibody. I will perform SDS-PAGE and I would like to detect a receptor in my sample and I don´t know whether monoclonal antibody will be better than polyclonal. Also, if I will use monoclonal antibody for my targeted protein, should I use even monoclonal antibody for my control protein? I also wonder if one these antibodies is more suitable for native electrophoresis. Thank you!
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nguyenmccarty
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Re: Western blot - choosing correct antibody
Hi! These are great questions to ask as you get started on Westerns!
In terms of a monoclonal versus polyclonal antibody, in general: a monoclonal will be more specific but less sensitive, while a polyclonal may be less specific but more sensitive.
Specificity: This is because a monoclonal antibody recognizes just a single epitope, or portion of your target protein, while a polyclonal antibody will actually be a mix of antibodies each recognizing a different epitope of your target protein. So if your target protein has portions that share significant homology (similarity) with other proteins (other receptors, for example), then a polyclonal antibody is more likely than a monoclonal antibody to bind those related proteins as well as your protein of interest.
Sensitivity: On the other hand, a polyclonal antibody can be more sensitive than a monoclonal because now multiple individual polyclonal antibody molecules can bind these distinct regions of your target protein, while monoclonal antibody molecules will have a more limited number of epitopes available to bind.
So the short answer to your question is use a monoclonal antibody if you can, but a polyclonal antibody may be better for you if, for example, your receptor of interest is not very highly expressed.
Regardless of whether you choose a monoclonal or a polyclonal antibody for your protein of interest, you can still choose either type to detect your control protein (GAPDH, actin, etc.). You are measuring that independently of your protein of interest, so how you detect one doesn't impact how you detect the other.
For native electrophoresis, you do need to choose your antibodies carefully, but not in terms of monoclonal vs. polyclonal. Many antibodies are raised against the denatured protein or even just a fragment of the protein, meaning the antibodies may detect portions of the protein that are typically not exposed (and therefore available for antibody recognition) when the protein is in its native form. On the other hand, if an antibody is raised against a native/properly folded protein, then the antibody may recognize a site that includes amino acids that would actually quite far away from each other if you were to unfold the protein. So if you perform native electrophoresis, you need to check on an antibody's product sheet whether it recognizes the native protein. In general for electrophoresis, denaturing conditions (such as SDS-PAGE) will suffice.
I hope this clarifies things for you, and good luck!
In terms of a monoclonal versus polyclonal antibody, in general: a monoclonal will be more specific but less sensitive, while a polyclonal may be less specific but more sensitive.
Specificity: This is because a monoclonal antibody recognizes just a single epitope, or portion of your target protein, while a polyclonal antibody will actually be a mix of antibodies each recognizing a different epitope of your target protein. So if your target protein has portions that share significant homology (similarity) with other proteins (other receptors, for example), then a polyclonal antibody is more likely than a monoclonal antibody to bind those related proteins as well as your protein of interest.
Sensitivity: On the other hand, a polyclonal antibody can be more sensitive than a monoclonal because now multiple individual polyclonal antibody molecules can bind these distinct regions of your target protein, while monoclonal antibody molecules will have a more limited number of epitopes available to bind.
So the short answer to your question is use a monoclonal antibody if you can, but a polyclonal antibody may be better for you if, for example, your receptor of interest is not very highly expressed.
Regardless of whether you choose a monoclonal or a polyclonal antibody for your protein of interest, you can still choose either type to detect your control protein (GAPDH, actin, etc.). You are measuring that independently of your protein of interest, so how you detect one doesn't impact how you detect the other.
For native electrophoresis, you do need to choose your antibodies carefully, but not in terms of monoclonal vs. polyclonal. Many antibodies are raised against the denatured protein or even just a fragment of the protein, meaning the antibodies may detect portions of the protein that are typically not exposed (and therefore available for antibody recognition) when the protein is in its native form. On the other hand, if an antibody is raised against a native/properly folded protein, then the antibody may recognize a site that includes amino acids that would actually quite far away from each other if you were to unfold the protein. So if you perform native electrophoresis, you need to check on an antibody's product sheet whether it recognizes the native protein. In general for electrophoresis, denaturing conditions (such as SDS-PAGE) will suffice.
I hope this clarifies things for you, and good luck!
Re: Western blot - choosing correct antibody
Hello nguyenmccarty, thank you so much for your great explanation! I think I won´t have a problem to choose the most suitable antibodies anymore after your advice!
I found a good antibody which according to a product sheet and 1 published article should detect my receptor in native form, so I´m thinking about to perform native PAGE instead of the SDS-PAGE. With the native PAGE I would be able to detect even whether my receptor binds its ligand, am I right? During native PAGE, the bonds between receptor and ligand stay intact, so I may see 2 bands on my membrane – receptor with and without ligand. Do I understand it correctly?
Thank you so much!
I found a good antibody which according to a product sheet and 1 published article should detect my receptor in native form, so I´m thinking about to perform native PAGE instead of the SDS-PAGE. With the native PAGE I would be able to detect even whether my receptor binds its ligand, am I right? During native PAGE, the bonds between receptor and ligand stay intact, so I may see 2 bands on my membrane – receptor with and without ligand. Do I understand it correctly?
Thank you so much!