Hi Samra,

All of these methods have their drawbacks. Also, you can actually measure BCA or Bradford on Nanodrop as well.

The thing is that both BCA and Bradford use a colorimetric reaction to quantify proteins in a solution. Then you can measure the absorbance using any spectrophotometer (e.g. Nanodrop). BCA is better for any detergent-containing samples but is prone to reducing agents (such as DTT). Bradford on the other hand is prone to detergents. Consult the composition of your sample and the table of interfering chemicals for each method. The table shows maximum allowed concentrations for most popular reagents. Choose the method that matches your sample composition best. In addition you can always dilute out the interfering reagent, as long as your protein concentration is still within the detection limit of the assay.

Results for both methods are affected to a high degree by aminoacid composition of the proteins in your sample. Meaning that, depending on which protein you will use as the standard (most commonly used I think is BSA), you might get different results for your samples.

Measuring by Nanodrop itself without any colorimetric reaction means you're just measuring the absorbance generated by aromatic aminoacids in your proteins, which again is highly dependent on the percentage of these aminoacids in your sample and how well the standard used reflects this percentage.

To summarize, if you're measuring complex protein mixtures (e.g. total cell lysate) and you just want to make sure you're loading equal amounts of protein in each sample on your gel use either Bradford or BCA. They're both convenient, BCA can be used in a very convenient high-throughput microplate format, which also saves your sample. Just match the method to your sample buffer composition.

If you're measuring purified protein and you know its aminoacid composition you could also use the Nanodrop measurement, just remember to choose a standard the matches your protein's composition best.

You can measure your protein after BCA or Bradford assay with Nanodrop. Check the next link:

http://www.nanodrop.com/protein.aspx

Hi Samra,

All of these methods have their drawbacks. Also, you can actually measure BCA or Bradford on Nanodrop as well.

The thing is that both BCA and Bradford use a colorimetric reaction to quantify proteins in a solution. Then you can measure the absorbance using any spectrophotometer (e.g. Nanodrop). BCA is better for any detergent-containing samples but is prone to reducing agents (such as DTT). Bradford on the other hand is prone to detergents. Consult the composition of your sample and the table of interfering chemicals for each method. The table shows maximum allowed concentrations for most popular reagents. Choose the method that matches your sample composition best. In addition you can always dilute out the interfering reagent, as long as your protein concentration is still within the detection limit of the assay.

Results for both methods are affected to a high degree by aminoacid composition of the proteins in your sample. Meaning that, depending on which protein you will use as the standard (most commonly used I think is BSA), you might get different results for your samples.

Measuring by Nanodrop itself without any colorimetric reaction means you're just measuring the absorbance generated by aromatic aminoacids in your proteins, which again is highly dependent on the percentage of these aminoacids in your sample and how well the standard used reflects this percentage.

To summarize, if you're measuring complex protein mixtures (e.g. total cell lysate) and you just want to make sure you're loading equal amounts of protein in each sample on your gel use either Bradford or BCA. They're both convenient, BCA can be used in a very convenient high-throughput microplate format, which also saves your sample. Just match the method to your sample buffer composition.

If you're measuring purified protein and you know its aminoacid composition you could also use the Nanodrop measurement, just remember to choose a standard the matches your protein's composition best.

Thanks Jose, Krzysztof and Gagaoua for your nice input. I am using RIPA lysis buffer for preparing the whole cell lysate so I think BCA will suit better than Bradford.

Yes, it would. That's what I was using in my previous lab as well and it BCA worked very well with RIPA.

I am having trouble in getting similar values for BCA and Bradford. BCA has lower amount whereas Bradford has 4x higher amount. Is there any reason for that?

' BCA has lower amount whereas Bradford has 4x higher amount. Is there any reason for that?'

Bradford assay detects the presence of basic amino acids viz lysine, histidine & arginine while BCA assay involves the reduction of the Cu2+ ion by cysteine, cystine , tryptophan & tyrosine. Consequently it depends on the relative abundance of the said amino acids in the given protein. Moreover, if there are interferents in your protein samples e.g salt, DTT, detergents etc, please recheck both methods with suitable controls that contain these interfering compounds alone (in the same concentrations as the test plus the reagents but not the protein samples)- this may give you a more accurate estimate.