Bradford Assays

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dmm520
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Bradford Assays

Is it possible to generate a standard curve with the bradford assay procedure using any protein?  Why would I use BSA versus the actual protein that I am trying to work with?

Thanks :)

Sami Tuomivaara
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dmm520,

dmm520,

It is possible to use any protein you want in generating standard curve, but because the colorimetric response factor of every protein is different, the best option is to use the same protein you are assaying.

In any case, you ultimately have to measure the concentration of the standard (whether BSA or your own protein) accurately. For this, you could weigh dried protein sample and prepare standard from that, or use UV absorbance (for this you need to know the absorption coefficient, it can be calculated, search for method of Gill and von Hippel).

People use BSA because lot of times you can't spare your precious protein to make a standard. Also, commercial BSA preparations are relatively pure and cheap, this way you can make a primary standard by weighing some amount of BSA (weighing method can be very accurate if you have analytical balance). And finally, because everyone else in the past used it, all experiments that have been standardized with BSA are then more or less comparable (in theory at least).

Cheers,

Add colour 2 ur life
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Hi,

Hi,
I am there with suola. Answer for your query is Yes,any purified protein can use it for standard prepartation not only for Bradford but also for any other method, if the protein is not so pure it might give some interference .People are using  BSA  for standard curve prepartation is because of its cost effectiveness,availability ,stability , reproducability (no variations from batch to batch preparation)and and so on.

All d best.

Balu...

dmm520
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Thank you for all of the help

Thank you for all of the help and insight ! I have been looking at various methods for protein concentration determination and I am wondering if UV spectroscopy is a better method than the bradford assay if dealing with a complex preparation and system of the protein (granted of course none of the other components absorb at 280 nm)...any thoughts? I have already tried a fluorescence method and this has given unreliable/variable concentration readings.

Thanks again!