Abstract
We present a strategy that overcomes the high background arising during Western blotting (WB) detection of proteins obtained through immunoprecipitation (IP). Traditional HRP-conjugated secondary antibodies, which detect the denatured heavy and light antibody chains, produce high background that often mask the signals of interest on WBs. Here, we show that HRP-conjugated Protein A and Protein G, which detect almost exclusively intact antibody molecules, can be effectively used to obtain clean and specific WB signals of target proteins.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, N.I.H., Intramural
MeSH terms
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Blotting, Western / methods*
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Horseradish Peroxidase / chemistry
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Immunoglobulin Heavy Chains / chemistry
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Immunoglobulin Heavy Chains / metabolism
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Immunoglobulin Light Chains / chemistry
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Immunoglobulin Light Chains / metabolism
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Immunoprecipitation / methods*
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Nerve Tissue Proteins / chemistry
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Nerve Tissue Proteins / metabolism
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Protein Binding
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Staphylococcal Protein A / chemistry
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Staphylococcal Protein A / metabolism
Substances
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G-substrate
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Immunoglobulin Heavy Chains
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Immunoglobulin Light Chains
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Nerve Tissue Proteins
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Staphylococcal Protein A
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Horseradish Peroxidase