Here is a compatibility table for several protein estimation methods. http://www.piercenet.com/files/TR0068-Protein-assay-compatibility.pdf

We were generally pleased with the BCA kit, as it was compatible with several detergents we use (

Dear Mamen,

DTT interferes with protein estimation, you can either choose to do dialysis or use Compat-Able protein assay preparation kit from Thermo Scientific (http://www.piercenet.com/instructions/2161308.pdf) to remove interfering substances prior to protein estimation. We use BCA Protein assay for Protein estimation it works fine for us.

Thanks

Have you tried Lowry's method? Only phenolic compounds interfere with Lowry's reagent.

I suggest to use photometric methods like measuring the absorbance of your sample under uv wavelength. Another way is to use biuret method.

First try Bradford method along with blank buffer. if result showing interferance. than go for precipiration with 72% TCA or chilled ethanol, dissolved pellet by using water or 2.5% SDS. than you can estimate protein by bradford or BCA method, both method will work fine.

According to a review paper that I read, the best method is biuret. I am sending the review, it is in portuguese, but it is not difficult to understand. In this review they show some tables with the interferent of each method. I hope it help.

Here is a compatibility table for several protein estimation methods. http://www.piercenet.com/files/TR0068-Protein-assay-compatibility.pdf

We were generally pleased with the BCA kit, as it was compatible with several detergents we use (

The Pierce 660nm assay is the best. Just add protein to the reagent and measure 660nM. I used in in purification of membrane proteins solubilized with different detergents. See the Pierce catalogue and read about it. It is also,compatible with reducing agents. I have used it with DTT, TCEP, and betamercaptoethanol.

It is cheap too.

As Marcia suggests, the 660 assay looks very compatible with all buffer components you mentioned, Mamen. However, the protein-to-protein variance is high by this method (up to 37%).

I think BCA method could do the job, but my preference would definitely be for the EZQ protein quantitation kit from invitrogen, a fluorescence-based protein assay that facilitates fast quantitation of protein samples prepared for gel electrophoresis. as the description of the kit says, this assay can be performed in the presence of detergents, urea and reducing agents. You need to simply spot 1 µL of your protein sample onto the prepared paper (in triplicate at least, so very small volume of sample required), then you have to stain the paper containing your samples and a calibration curve with a fluorescent dye, and then measure the fluorescence. The kit comes with 96-well microplate and is suitable to be used in a microplate reader or a laser scanner.

Good luck!

I did a comparisson of Bradford and BCA for plant proteins to see which method is more accurate. Both assays are quite stable but the most important difference is that: 1.) Bradford measures on a protein level (mainly binds to arginine, tryptophan, tyrosine, histidine, and phenylalanine) 2.) BCA measures already tripeptides. Consequently, with the BCA you might measure higher content due to some protein degradation, which might occur in stress Experiments (maybe not the case with muscle cells?!). Further, the BCA is more sensitive, which means you can measure higher concentrations. But this is actually no problem because you can always down dilute you sample. If it is mainly a question of interfeence with detergents or reducing agents, you can find the interfering chemicals easily on the internet (eg. http://www.labome.com/method/Protein-Quantitation.html).

Good luck with your quantification.

In majority of assays referenced above, the read-out will truly depend on the concentration of detergent you are using. Lower concentration of detergents would be compatible with many assays above without sacrificing the quality of lysis. Alternatively, you could always avoid time-consuming colorimetric methods and simply use nanodrop or as suggested above spectrophotometer.

My experience is that detergents at doses of 1% or less have little chances to interfere with the protein assays suggested. To be sure, you can try to compare the protein content of your samples before and after an oil extraction (soybean, castor oil) that can remove 80% or more of non-ionic detergents like Triton or Tween, without extracting proteins. Alternatively you can run it (especially if small volume) through a pre-packed C18 column that will adsorb the detergent, without adsorbing the proteins, unless they are somewhat hydrophobic

The specs for the reagent you purchase will have the info. But I know from using the Pierce 660, that as Thierry has stated, that with detergents such as NP40, CHAPS, dodecylmaltoside, Triton X 100, that for 1% or less, this reagent is not a problem. But you can get the specs online.

Mamen, I would follow a completely different approach. If you r trying to run IEF with the proteins that you are extracting with this buffer you will probably get a not very good focusing unless you do a protein precipitation or a liquid/liquid extraction. Once you have your protein pellet in acetone, you can use BCA for quantitation

I think Bradford assay will work in such cases. If you need accurate estimation do a TCA precipitation and then use Lowry method

Siva opens up a new query on methods for pptg proteins. TCA pptn usually gives variable yield of your protein (we found only 35-60% recovery with BSA). Acetone pptn is better. Perhaps PEG maybe better.?

As I understand TCA can precipitate most of the proteins but the only problem sometimes is that it denatures the proteins making it difficult to resolubilize . I may go with acetone but not with PEG

We could resolubilize BSA from the TCA ppt easily--but the yield was low (repeated thrice). Precipitation is easy. Recovery of your protein after pptn is the problem. For a given protein, One may have to compare different pptg agents before choosing one.

You can please refer to the links below to obtain certain interesting and valuable informations:-

1. http://www.bio-rad.com/LifeScience/pdf/Bulletin_1069.pdf

2. http://www.genotech.com/bulletins/protein-assays-introduction.pdf?ref=binfind.com/web

We have tried Biorad protein concentrate dye reagent for protein estimation in extracts obtained with a lysis buffer having urea, thiourea, chaps and DTT and it worked. Rehydration buffer is taken a s a blank with this. Morever, method is supplied alongwith the reagent.