Difference between revisions of "20.109(F08): Mod 2 Day 5 Probe western, isolate RNA"
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Antibodies are useful tools in the lab. Today we'll use antibodies to detect a protein on a blot. This technique, called Western analysis, can give us information about the size and concentration of the protein in the pool that was separated by SDS-PAGE. In our case, we'll use a Western to characterize the TAP-tagged proteins we've generated. Hopefully you're now confident that you've modified the gene of interest with a TAP tag sequence, but you could imagine a case where the genome has the correct TAP-TRP sequence and yet the resulting protein is not seen on a Western blot. Perhaps it's unstable or expressed at such a low level that it's not detectable. Alternatively, you could imagine that there are cellular proteases that might cleave the TAP-tag off the protein we've attached it to, ridding the cell of the fusion we've tried to make. Both of these outcomes are possible (even common!), and would be critically important pieces of data to gather before performing a TAP purification of the tagged complex. Assuming the protein we're interested in is expressed and intact, then in general, the quality of Western results depends on the quality of the antibody we choose. | Antibodies are useful tools in the lab. Today we'll use antibodies to detect a protein on a blot. This technique, called Western analysis, can give us information about the size and concentration of the protein in the pool that was separated by SDS-PAGE. In our case, we'll use a Western to characterize the TAP-tagged proteins we've generated. Hopefully you're now confident that you've modified the gene of interest with a TAP tag sequence, but you could imagine a case where the genome has the correct TAP-TRP sequence and yet the resulting protein is not seen on a Western blot. Perhaps it's unstable or expressed at such a low level that it's not detectable. Alternatively, you could imagine that there are cellular proteases that might cleave the TAP-tag off the protein we've attached it to, ridding the cell of the fusion we've tried to make. Both of these outcomes are possible (even common!), and would be critically important pieces of data to gather before performing a TAP purification of the tagged complex. Assuming the protein we're interested in is expressed and intact, then in general, the quality of Western results depends on the quality of the antibody we choose. | ||
− | Luckily a high quality antibody can have a relatively low affinity for its target protein and still be useful for Western analysis, . This is because the target is localized and concentrated on a blot, allowing the antibody to bind using both antibody | + | Luckily a high quality antibody can have a relatively low affinity for its target protein and still be useful for Western analysis, . This is because the target is localized and concentrated on a blot, allowing the antibody to bind using both antibody |