Hey all
A few of my lab-mates store the protein after quantification (I used bradford assay) in cell lysis buffer containing protease inhibitor in -80. While others add loading dye and denature the protein in 95 degree and store it in -20.
I have quantified the protein in the cell lysate. However dye to time constraints and huge sample protein I couldn't add the loading dye after the quantification process. (2 hours had already passed While I was calculating the amount of loading dye required for each sample). I got panicked thinking the protein in the cell lysate would be degraded and hence upon an advice from a fellow senior I aliquoted 20uL of each sample into another 1.5 mL centrifuge tube and stored the the stock and the aliquot in -80. So that I need not freeze and thaw my stock again and again.
Following are my queries
1. At what stage is it recommended to store the protein?
2. Does the concentration differ after storage?
3. Do I need to do bradford assay once again after I thaw them from -80?
4. what is the incubation period for bradford assay? (after adding BSA to the bradford reagent how long should I wait to take the reading or should I take the reading straight away?
Thank you
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I think it was a good idea to set aside a small portion of the lysate for subsequent quantitation and preparation for electrophoresis if you were not able to do those steps immediately. I also agree with storing the bulk of the lysate at -80oC if you can't immediately proceed with purification steps. However, it would be better to prepare the lysate when you have time to go directly to the first purification step, since the lysate is the point at which proteolysis is most likely to be a problem. Freezing should be done as rapidly as possible, using a dry ice/ethanol bath or liquid nitrogen.
The concentration should not change during storage, unless there is significant precipitation, so it should not be necessary to repeat the Bradford assay. Make sure the sample is well-mixed after thawing, because some separation of solutes can occur during freezing.
The incubation period for the Bradford is 5-10 minutes. If you wait too long, the protein will precipitate due to the acidity of the reagent.
Thank you dear Adam B Shapiro for your insights and answering my queries.
Hello,
I typically store my protein sample in the cell pellet directly after protein production. So far I haven't had any problem recovering my proteins this way using BL-21 as the heterologous host.
Otherwise I would complete the lysis and purification steps before storing the protein at -80. The amount of protein won't change after this step, but the activity may decrease especially on repeated freeze-thaw cycles, so quantify your protein and divide it into single use aliquots.
Also, if you want a faster quantification method, you can calculate the extinction coefficient, measure the absorbance at 280 nm, and use this formula to find your protein concentration:
concentration = absorbance/extinction coefficient * path length
It won't be as accurate as the Bradford assay, but it's considerably faster and uses less protein. I use this site to find the extinction coefficient: https://web.expasy.org/protparam/
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