I am working on the isolation and caracterization of extracellular vesicles content by western blot technic.
But I am not able to get for each experiment good concentrations for the EVs fractions. I said maybe it can be due to the fact that I only take 1uL of sample diluted in 24uL of water. Maybe I loose material and it's not accurate enough.
Could you please recommend me other protocols for protein quantification?
Thank you
This is the protocol I use for the Bradford assay. It can measure down to about 0.5 µg in 10 µl (0.05 mg/ml) easily. By increasing the sample volume, it should be possible to make it more sensitive. Pre-concentrating the sample by ultrafiltration or precipitation would also increase the sensitivity.
The sample in 10 µl is added to a clear 96-well assay plate with flat-bottom wells. Bovine serum albumin standards are also added in 10 µl. I use standards that include 0, 0.5, 1, ..., 5.5 µg of BSA, but for dilute protein samples, it would be better to use lower amounts of BSA for the standards, such as 0, 0.2, 0.4, ..., 2.2 µg. The samples should not contain anything that interferes with the assay, especially detergents. Two hundred µl of Bradford reagent from Bio-Rad Laboratories is added to each well. After mixing the plate on an orbital plate shaker, wait 5-10 minutes and measure the absorbance at 595 with a plate reader.
https://www.bio-rad.com/featured/en/bradford-assay.html
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