I am doing a BCA assay to determine and then standardize the protein concentration of my samples. The goal is to be able to use an aliquot from a sample tube
to determine its concentration, so that I can infer the amount of protein present in that sample and standardize all the samples without counting worms.
Problem: we do not have a homogenizer or sonicator in our lab that can be used on worms. To break up the worms I have tried the liquid nitrogen/heating method and am currently using base/heating method (NaOH + 20mins at 70C). I find that
the latter works really well, but I still get a few small worm chunks floating around in solution. I try to get around this as best I can by pipetting quickly up and down.
However, there are still some small chunks left, and I am concerned that I am not estimating protein concentrations correctly. Can anyone advise on 1) if this is okay as is because the # of chunks compared to the whole worm extract is so negligible and small 2) if this is not okay and recommend a way to get rid of the worm chunks and to get them to go into solution and dissolve completely without buying an expensive homogenizer or sonicator.
I’d recommend centrifuging your sample to remove the leftover chunks and then proceed from there. In the following link where the authors analyze age-dependent proteomic changes, they removed carcasses by spinning at 3000g following lysis: http://www.ncbi.nlm.nih.gov/pubmed/22103665
Thank you. Will give it a try. Though my main concern has been that I am underestimating the protein content by getting rid of the chunks rather than having them go into solution (i.e. each tube has a slightly different number of the small chunks and if I get rid of them, that will affect the protein concentration differently in each of the tubes - since goal is to use protein concentration in each tube to estimate original protein concentration in said tube rather than get the same amount/concentration of protein from each tube after the fact).
Sorry for the delayed reply. So the goal for this is the BCA Pierce protein quantification assay. I want to ensure equal loading into each well of a 96 well plate without having to pick worms for a fluorescence assay. I load those wells from tubes, and reserve some worms in each tube, then BCA kit the remaining worms to back calculate the original amount of protein I loaded into each well of the 96 well plate (i.e. I standardize how much material goes into each well by protein concentration rather than by worm number). My concern is that in those little clumps that get left behind, I am losing some protein, and so that when I use an aliquot for the BCA kit assay, it is not accurate because those small clumps do not go into solution, and so I am underestimating the amount of protein. And while it could be argued that I am underestimating in the same way equally across all tubes, since the clumps are not uniform, I don’t think that’s the case. Then again I don’t know if it’s a serious enough problem to throw off my readings. Mostly when people do this, they first get the protein concentration and then load from the tube with a known protein concentration into a Western, etc. I am sort of doing things backwards for this assay, so think that I need to have everything go into solution. If I just centrifuge it out, that still doesn’t get at the underestimation problem.
Steve, thank you for the hand-held homogenizer tip. I’ve been searching for something like that that is sturdy for a while, and may just give it a try.