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1. Review and analyze the data (from all five groups) for part A. Here are some questions you'll need to address.
There was no obvious pattern to the data, as all of you pointed out. Ltv1-His was the only construct that was detectable in all three lanes, and Yar1-S was the only other construct that was detectable at all. Interestingly, Ltv1-His has 44 lysine residues, while the other four constructs we're using have 15-18 each. Since lysines are the only residues that can be tagged with biotin in this method, this may explain why Ltv1-His is the most readily detectable product. However, the beta-gal positive control has 62 lysine residues per molecule, and yet was undetectable in both cases (although only 4 microliters of this control were loaded per lane).
Clearly our detection method was not sensitive enough for our purposes. My attempt to optimize this by reducing the blocking time from overnight to 30 minutes was not successful--I detected no signal in any lanes. (It's not clear to me why I was unable to detect even Ltv1-His.) Further optimization is necessary.
2. Review the protocol for part B.
(2 pts) What proportion of each flow-through fraction was loaded on the gel? (In other words, what proportion of the proteins that were added to the beads--but did not bind to them--was loaded as the "flow-through" sample in each case?)
The key here is to focus on the proportion of the proteins that were added to the beads but did not bind to them. 250 µl of lysate was added to each bead aliquot. After the binding reaction, the flow-through was removed. There was 250 µl - 275 µl of flow-through, depending on whether you take into account the 25 µl of lysis buffer that was already present in the bead aliquot. Of this, 5 µl was loaded on the gel (half of your 10 µl aliquot was flow-through, and half was sample buffer). Therefore the proportion that was loaded to the gel was 5/250 to 5/275, or 1/50th to 1/55th.
It is not relevant that you only saved 50 µl of flow-through and discarded the rest. What is relevant is the total flow-through volume.
(2 pts) What proportion of the proteins that did bind to the beads was loaded on the gel?
Your beads were boiled in 50µl of sample buffer, a procedure that should result in all of the bound protein dissociating from the beads and entering the sample buffer. The total volume of bead-bound protein is therefore 50 µl, of which you loaded 10µl, or 1/5th.
(2 pts) Given the above information, look at the data from part B (for all three groups). Does it appear to you that GST or GST-Yar1 bound quantitatively to the beads? (Did most or all of it bind?)
You loaded 10 - 11X more of your bead-bound protein than of your flow-through protein, and yet the intensities of the bands were similar. If anything (especially for GST-Yar1), the bead-bound fraction produced a fainter band. The binding was therefore not quantitative--10% or less of each protein bound to the beads.
(2 pts) Given the data, how specifically did GST and GST-Yar1 bind to the beads? (How pure does the bead-bound fraction appear to be?) I'm not looking for a number here, just a qualitative judgement.
Although it's not obvious from the scanned gels (and you therefore didn't have to point this out) there are some faint background bands in each bead lane. The binding is therefore not 100% specific, although it's pretty good. The fact that it is not 100% specific means that we'll have to be careful in interpreting any binding of in vitro-labeled protein to the beads. We can still interpret our data (if we can detect any...) but we'll need to be careful about our wording.
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Created by:
bkbaxter@lclark.edu
Updated: 24 November 00