Hi Joe,

since the Bio-Rad protein assay seems to be some kind of Bradford assay the high amount of detergents (SDS, Deoxycholate, NP-40) will interfere pretty much with this kind of assay. This is due to the binding of detergents to the proteins on the one hand and binding of the detergent to coomassie on the other hand.

Check the manual for the critical concentrations of these detergents! On option would be to dillute your samples and the "blank" to get below these concentrations interfering with the assay.

I use a modified Lowry method for the determination of RIPA protein concentrations. First I precipitate the proteins with TCA and redisolve them in a buffer without detergents. I can send you a protocol if you want.

If you are more into "kit-science" I would recoment the BCA-protein assay, since it is pretty indepentend of detergent in your buffer, since it is based on the reduction of copper.

Best,

Kai

I do not know exactly what you mean the color change. I use RIPA or MOPS as blank for protein assay but have not really experience similar problem. But, the color does change if I let the plate sit several hours or overnight. Sometimes I can see slight color change due to the background or somebody spills protein containing solution into my 96-well unused portion. My students like to use my plates for unknown reason and I had a lot of this kind of issue. Usually I just get another new plate to measure again. Sorry I can not give you a good suggestion.

Hi Joe,

since the Bio-Rad protein assay seems to be some kind of Bradford assay the high amount of detergents (SDS, Deoxycholate, NP-40) will interfere pretty much with this kind of assay. This is due to the binding of detergents to the proteins on the one hand and binding of the detergent to coomassie on the other hand.

Check the manual for the critical concentrations of these detergents! On option would be to dillute your samples and the "blank" to get below these concentrations interfering with the assay.

I use a modified Lowry method for the determination of RIPA protein concentrations. First I precipitate the proteins with TCA and redisolve them in a buffer without detergents. I can send you a protocol if you want.

If you are more into "kit-science" I would recoment the BCA-protein assay, since it is pretty indepentend of detergent in your buffer, since it is based on the reduction of copper.

Best,

Kai

Thanks Kai,

So I was diluting the samples all 1:10 but I would also dilute the blank, i.e. the RIPA buffer 1:10 as well as you suggested. This has been working pretty well for me so far. However your precipitation protocol seems interesting, I would love to take a look at it!

Thanks,

Joe

I use RIPA containing 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0. No changes of color observed with Bio-Rad protein assay (Bradford). Thus the simplest explanation for me is contamination of your RIPA with protein...

To check it precipitate your RIPA with TCA or equivalent and run on the gel. Another option is to verify your RIPA from the new/freshly made stock.

Best - Jan

For RIPA buffer, I would recommend using BCA method, such as the kit provided by Pierce.

Good luck.

Xiaonan

I agree with Kai, it is highly likely that there is some ingredient(s) within the RIPA buffer that you are using that are not compatible with your Bio-Rad protein assay. Most protein assay’s come with a list of compatible substance concentrations; I would work out what is in your RIPA buffer that is causing the problem.

In the past I’ve used RIPA buffer with the BCA assay (Pierce) and had no problems.

Best Wishes

Manda

Hi Joe,

I dilute my standards/samples in the RIPA buffer used to extract my protein and then use the same RIPA buffer as a blank. I then take the blank O.D. away from the O.D. for my standards and test samples, accounting for any interference from the RIPA buffer.

SDS and Detergents (NP-40 or Tween or Triton-100) change the background color of the bio-rad protein assay reagents. Basically you substract the blank sample (only RIPA) with the protein sample. Make sure that you perform standarization curve properly taking in to consideration of the factors.

Easy fix, BioRad has a solution "S" you add to Reagent "A", I believe it is 20ul of "S" to every ml of Reagent "A" So they are compatible.. I've been there, done that & fixed it.

I also used RIPA with BCA Kit from Novagen, no problems had I. It seems like a contamination problem.

If you see a mild or slight color change, just subtract it from all, which you are supposed to do with a blank always. But if you see an extensive color change with the blank, which means your RIPA buffer is heavily contaminated, your standard might become unreliable, then don't use it.

As others have previously said, the Bio-Rad assay is basically a Bradford assay and it is well known and documented that detergents (as well as many other chemicals) can cause high backgrounds. You can dilute as suggested if your starting protein concentration is high enough to allow dilution and still get a signal in the diluted samples or you can run blanks (as others suggest) but the accuracy will depend on the signal to noise (protein vs background).

If you are running Bio-Rad assay it comes with a long list of reagents that cause background interference I am surprised no seems to read this information. I have run Bradford assays since the early 80's but switched to BCA about 15 years ago due to the incompatibility of the Bradford reagent with other chemicals commonly used in protein extraction and isolation/purification.

As suggested above you can precipitate protein with acid (usually TCA) spin and wash but if you are really trying for quantitative results you should include a known protein amount as an internal control to look at recovery of protein as it is typically not 100% doing TCA extraction by ether or neutralization.

Hi,

If your RIPA buffer has NP-40 in it, it does react with the Bio-rad protein assay. I would dilute the RIPA same as 1:10 as samples for more accurate reading, however, if your concentrations are low, between your 0 reading and your lowest two BSA controls, those readings are typically not accurate and actually lower than the reading you will get. This is why if you do use NP-40, you should use the Pierce BCA assay instead as it doesn't react with NP-40.

Hi,

I often spike my standard curve with equivalent volumes of RIPA buffer to compensate for this. For example, if I make up 80ul of a standard sample, i replace 4ul of the diluent (eg distilled water) with RIPA buffer (a 1:20 dilution). I also dilute my samples 1:20 so there is 4ul of RIPA buffer (containing my samples) in a total of 80ul diluent (eg distilled water). Obviously volumes can be adjusted for any dilution.

Hope this helps.

hi

i agree...due to some of RIPA Buffer including sodium deoxycholate as well as the reducing agent like DTT added before use, i have generally found an interferance in the bradford assay...BCA assay works very well with RIPA buffer and is free from any unnecessary colour development.

We use a different protein detection kit from BioRad called the DC Protein Assay (inferred by Kellie). It is essentially the Lowry Assay. We routined use RIPA for our cells and have had no issues.

Tris-based buffers can also interfere......most protein assays are sensitive to Tris above a certain concentration. Try diluting your sample in a buffer without Tris to lower the Tris concentration. Use the same dilution to make up all your standards as well. Also make sure that your assay is SDS compatible. Bio-Rad makes an assay that used an additive to chelate SDS and therefore not interfere with the protein assay reagent(s)

I got the same problem and I solved it using BCA.

Good luck

I sometimes use the Bradford assay with RIPA and compensate by making up the protein standards (e.g. BSA) freshly in RIPA as well. This works OK but the dynamic range of the assay is reduced due to the detergent, making it useful for rough estimations only. For more accurate results I'd use the BCA assay as many other people suggest, although the kits are quite expensive...

I disagree that BCA is much more expensive that biorad kit if you are following data sheet recommend:

biorad, microplate of 1 mL

110$ -> 450ml -> 200µl per reaction -> 2250 reactions -> 0.048$ per reaction

BCA, microplate of 200µl

160$ -> 500ml -> 162µl per reaction -> 3086 reactions -> 0.052$ per reaction

the 162µl comes from

1 volume of solution B in 49 volumes of solution A -> for 200µl -> 196µl A + 4µl B

1 volume sample in 7 volumes of BCA -> for 200µl -> 162µl BCA 28µl samples

I would rather say that the biorad kit is really fast (5 minutes incubation and let's measure) and that the BCA is less sensitive to detergent and more sensitive (but need to be incubated at 37°C for at least 30 minutes)

But if you consider to use only 200µl of the biorad kit (will be difficult for the dilution of samples), the BCA kit is 5x more expensive... but still stay 0.05$ per reaction, so less that the price of a reaction tube.

Hi,

I proposed to read the below article:

Quantitation of protein.Noble JE, Bailey MJ.

doi: 10.1016/S0076-6879(09)63008-1.Methods Enzymol. 2009;463:73-95.

I hope to solve your problem.

We also use the DC protein assay to quantified our proteins and works well with RIPA Buffer. Try it!!!

RIPA buffer and Bio-Rad protein assay for me but some of my colleagues prefer BCA.

It's really a matter of "taste" but I think RIPA and 49:1 Biorad is easy and gives you what you need unless you really need a very accurate protein measurement. If you need that then I'd definitely go for BCA.

Hi Joe,

please try out 660nM Pierce protein quantitation assay supplemented with a pack (or more as needed) ionic detergent compatibility reagent if you're using RIPA plus Triton-X100 lysis buffer. Works miracles in 5 minutes, no need to wait for temeperature-sensitive and time-consuming results of BCA. I never trust Tris-HCl-based lysis buffers for colorimentric assays, and always make my own RIPA for tissue lysates:

25 mM HEPES (PH 7.4)

150 mM NaCl

1% Triton-X-100

0.1% SDS

0.5% Sodium deoxycholate (SOD)

5% glycerol

2 mM EGTA (not EDTA)

1 mM DTT (optional)

After spinning the lysates down I transfer the supernatant to different tube, and lyse a pellet in a smaller amount of the same buffer, except NaCl is 300 mM and it's supplemented with 70mM n-octylglucoside (releases lipid-raft-associated proteins). Yes, SOD alone is not enough to release lipid-raft associated proteins.

Then I centrifuge the tube on max, mix the supernatants, and then measure protein content.

You can simply use DC-Protein Assay from Bio-Rad... (DC = Detergent Compatible)

Yes, the detergent in RIPA will change the color of BioRad Protein Dye. If your samples are not that diluted, I just tried, 40 times dilution of RIPA won't affect the color reaction between proteins and the Protein Dye.