Problems with Bradford assay - (Mar/28/2006 )

Hi everyone!

We are using The Bradford-Assay from BIORAD to get the concentrations of our proteinlysates. If we fill the solutions to our lysate, we have to wait for nearly 15 min and the solutions has to be stable for an hour, but it isn't. The concentrations are not stable for this time. After a while we have to ReBlank and after this we get different concentrations of the same samples.

Can anybody tell me how long the Bradford is stable and how we can realize the ending? Has anybody the same experiences?

Thanks a lot

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light and temp sensitive. address these issues, then try a different system.

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light sensitive system in addition to time. try to work within your time frame parameters or look for a
different system. biorad is not the best.

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Can you be a little bit more specific as to how u do the bradford assay?? I used this all the time and I do not wait for 15 mins or 1h for anythng to stabilize...

Can you elaborate on your protocol??

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Hi, i've been using Bio-Rad kit protocol. Check if you use the right kit because they have different type for different protein lysis solution ingredients. Maybe you should consider this.

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Hi,

Compare your lysis solution ingredients (CHAPS etc) and use a compatible kit from BIORAD. I agree they have many different products and you need to select the best one for you.

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Hello everyone!

thank you for your help!

We use RIPA buffer to lyse our protein. The solution we use is as easy as it can be. Take 1µL Proteinlysate and give 1mL solution into a cuvette. Wait 15 min reaction time of the solution with the protein and measure...

In this time I have to go in another lab because our spectrometre is out of order
The solution is in this time on ice in a dark chamber...

any idea whats wrong??

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are you using the standard or the micro method?

if the standard method, have you filtered the diluted reagent?

we use the micro method in microfuge tubes and find that the dye-protein complex tends to be adsorbed onto the tube wall so the readings will reduce over time. to combat this we use timed addition and subsequent reading of the samples and blanks. we also try to run the samples in increasing protein amounts. when we have to read down, we rinse the cuvette with a used blank to remove any protein coating that may have formed.

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I also use RIPA buffer and due to the Triton X-100 i had a lot of interference with bradford. I changed to the BCA assay (Pierce) and it works great.
If you want to continue with Bradford i would dilute everything at least 10 times. I used to do that and that gave moderately reproductive results. But the BCA assay is easier and less work.

good luck

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I would say dont worry about the nitty gritty details. If you are doing a western then make sure you do the same each time. Even if you are making an error...the error gets distributed evenly throughout all the samples and you will end up having the same amoutnt of protein in each well.

I agree with someone who said earlier about the 10-fold dilution wwith RIPA as SDS alone gives a color change...but make sue you r consistent.

Also have a standard with known amount of BSA in RIPA. this will help you out too...

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