hi, I need to know whats the diff between individual ip and western with that particular ab?
If I need to check expression of one subunit of large five member complex like in my study p50 of nfkb then whats the diff if I check with ab specific for p50 with western or individual IP?
There is another difference between IP and Western blot. For IP, you typically need an antibody that recognizes the target protein in its native fold. In its native fold, however, the epitope your antibody is recognizing may be sterically obscured (if the antibody was merely raised against a short peptide sequence). Hence, many antibodies that work great for Western blotting do not work for IP. Moreover, if your protein is part of a large five member complex, as you say, it is possible that one of those five members might bind your target protein exactly or very close to where your antibody binds its epitope. Thus, again, access to this epitope might be sterically blocked and your antibody might not work. In contrast, in Western blotting the target protein is typically completely denatured (due to the SDS). Thus, antibodies raised against a short peptide should recognize their (denatured) target protein, even if that short peptide is not accessible as long as the protein is natively folded. However, many antibodies are not raised against short peptide sequences but against folded protein. Since SDS-PAGE denatures the protein, these antibodies will then not work for Western blotting (but may work for IP instead).
Long story short, it really depends on the type of antibody you have, what epitope it recognizes, and whether the epitope is conformation-sensitive.
There is another difference between IP and Western blot. For IP, you typically need an antibody that recognizes the target protein in its native fold. In its native fold, however, the epitope your antibody is recognizing may be sterically obscured (if the antibody was merely raised against a short peptide sequence). Hence, many antibodies that work great for Western blotting do not work for IP. Moreover, if your protein is part of a large five member complex, as you say, it is possible that one of those five members might bind your target protein exactly or very close to where your antibody binds its epitope. Thus, again, access to this epitope might be sterically blocked and your antibody might not work. In contrast, in Western blotting the target protein is typically completely denatured (due to the SDS). Thus, antibodies raised against a short peptide should recognize their (denatured) target protein, even if that short peptide is not accessible as long as the protein is natively folded. However, many antibodies are not raised against short peptide sequences but against folded protein. Since SDS-PAGE denatures the protein, these antibodies will then not work for Western blotting (but may work for IP instead).
Long story short, it really depends on the type of antibody you have, what epitope it recognizes, and whether the epitope is conformation-sensitive.
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