I need to measure the brain tissue protein concentration, but the tissue was homogenized in 90% methanol, then mixed with same volume of acetonitrile and spun down. 2. How can I get higher extracellular vesicle yields from cell conditioned medium? Does bromophenol blue affect the BCA assay? To compare the results of total mass (ng QCM) and concentration (µg/ml BCA Assay) I calculated both to µg/cm². You can confirm your results by silver staining of your gels. I have seen some muscle papers that work at a 1:50 and 1:100 ratio, but the product insert shows a 1:8 and 1:20 ratio. My goal is to calculate a ratio of specific protein to overall protein from these samples. Direct protein estimation from cultures cells in a 96 well plate? Open Biomed Eng J. If your samples contain both detergents and reducing agents, Try the Pierce 660nm. I am looking at cytokines levels of lungs exposed to an allergen. Unfortunately the blank as well as all the standards (using CDCHO as a diluent) gave very high readings, often bigger than my actual secretome samples. I'm at a loss and any help or advice you could offer would be appreciated! Why do I have trouble in quantifying protein extracted from Neuronally differntiated Neural Stem Cells with BCA assay? t-Octylphenoxypolyethoxyethanol, Figure 2. however, the bands could be seen on both sides of the 0.45 um nitrocellulose membrane. I have gone through the paper which you linked for the modified Bradford assay. I wonder what should I change while working with Jurkats. A table for compatible buffers/vompounds with the BCA method is attached. We did the Pierce BCA assay and ran the standard curve (and were able to estimate protein concentrations in our samples). Thanks for the comassie suggestion as well, I suspect you might be right about the degradation. Could be that your enzyme is not alone and that fraction 29 contains contaminating proteins or misfolded and enzymatically inactive Taq. After I decided to formaldehyde cross-link, my protein quantification has showed significantly less protein. PS: This doesn't happen all the time. The BCA Protein Assay combines the well-known reduction of Cu 2+ to Cu 1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu 1+) by bicinchoninic acid (BCA). We already tried Tween-80 of different manufacturers and grades, all with the same result. How to calculate sample protein based on standard protein curve in BCA? Lauryl sulfate sodium salt, I think precipitating the protein to get rid of contaminants is a good idea. The linear regression will also ensure proper weighting of the data points. How do I quantify protein from lipid rich liver tissue? We are trying to quantify protein encapsulated in liposomes. Before I repeat this assay, can anyone tell me does Optiprep reduce absorbance values in a protein assay? If in doubt whether they survive, use a viability marker to stain the cell suspension. Did u end up using it ? here X is the unknown protein concentration. I do not recommend to replace CHAPS/TBS with RIPA, as lysis/extraction conditions are different and therefore, it might affect  yield/purity of corresponding fractions. I have tried various methods for isolating these vesicles such as the ExoQuick-TC commercially-available reagent from SBI as well as notable centrifugation methods like: Lässer, C., Eldh, M., Lötvall, J. I've been using microBCA for protein concentration estimation for exosome samples. er~~I am confusing :( thank you for your kindly suggestions in advance:). INTRODUCTION AND OBJECTIVES METHODS Expression and Quantification of GFP Protein. All the values were accompanied by the statement ''The coefficient of determination for the regression evaluation of the standard series is < 0.8''. What I observe more than three times is that the signal of RIPA:PBS 10 times dilution is lower than the signal of RIPA:PBS 100 times, which suggest the RIPA actually is not increasing the signal right?