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Groundhog Day in Protein Purification
Submitted by Jennifer Newsted on Sat, 02/23/2013 - 11:13am
Why are Bradford assays like Groundhog Day? You'll have to read about it to know.
Apparently I’m in movie simile mode this week too because this week was like being stuck in the movie Groundhog Day. We started our week with the Bradford assay. I made serial dilutions from our first aliquot. After checking with some other lab groups, we determined that we would have within range results using 1/10, 1/20, 1/30, 1/40, and 1/50 dilutions. Next we mapped out our 96 well plate to run our assay in duplicate. We got a beautiful standard curve with an r^2 value of 0.991. Then we discovered that because we only ran our standards once we couldn’t use our results. So then it was back to the (Bradford) drawing board.
The next day when we came in, I made more dilutions. This time, I spoke with another classmate, and decided I liked her dilutions better. So I made 2 sets of dilutions (mostly for the practice since they were really close to each other). The first set as before. The second set were ½ dilutions from ¼ to 1/64, this time, using aliquot 2. Once again, we mapped out our plate and my lab partner loaded it up using duplicates of both sets of dilutions. Then we got our results. We decided the dilutions had to be off because they weren’t making sense. Dilutions that should have been weaker were stronger than their duplicated counterpart. Also, the variances between what should have been relatively close dilutions like 1/8 and 1/10 were too far off from each other for our comfort.
Groundhog Day number three (aka Wednesday). We come in. We make dilutions. This time, we ditched the ¼-1/64 dilutions and went back to our 1/10-1/50 dilutions. This time, my lab partner did the Bradford and I tackled the beta-gal assay. The beta-galactosidase assay measures the activity of the enzyme beta-galactosidase by measuring the reaction when ONPG is cleaved into the substrates ONP and galactose (ONP and G). After calculating how much Breaking Buffer/DTT I need for the assay, I prepared the solution adding the required amount of DTT to the already prepared breaking buffer (which we used in our dilutions). One beta-gal assay later, I misinterpreted my results. After using the equation to determine how many active units of enzyme we had per mL, I thought those numbers should be fairly similar once they’re multiplied by the dilution factor. This made me think that somehow my dilutions were off again. Later (Friday) I found out from our instructor that isn’t necessarily the case. Ouch. Well, at least I’m getting a whole lot of practice.
Long story short: Thursday we believe we were the victim of a warped plate on the Bradford and I got a decent beta-gal assay. Friday we finally got results that made sense on the Bradford assays that my lab partner and I both ran separately; though not until after I programmed the template for the plate reader incorrectly the first time and ended up with an r^2 value I hope I never see again so long as I shall live. My standard curve graph was actually inverted. Life lesson learned. Don’t incorrectly program the template! After the initial shock wore off my lab partner and I got a good laugh out of it.
Lessons of the week:
1) Always run your standards in duplicate.
2) Always run your blanks in your standards columns.
3) Always run your samples in triplicate (so you still have an average in case there's an outlier).
4) Load the plate reader template carefully. Especially when you skipped a row because of a known pipetting error.
5) When it makes no sense, ask your lab instructor and then do it again. And again. And again possibly.
Next week we move on to dialysis and ammonium sulfate precipitation. I'm really enjoying protein purification. The different characteristics of certain regions of the protein being used to help separate that particular item you're looking for is absolutely fascinating to learn about.
Feel free to peruse my website for protocol information at jennifernewsted.wix.com/biosciencetech!