Bradford Assay Questions

[ishbar]

Hello, I'm new here (obviously).

 

I'm a Psychology major, with a BMS minor, attending Central Connecticut State University. I figured that if I wanted to help remember some of the jargon in relation to my minor, it would greatly benefit me to emerse myself into any kind of science environment--such as these forums! I'm a Junior at the moment, however I have found some of my sophomore level classes can be quite trying--such as BMS 201.

 

Right now the topic we're covering is Bradford Assays (since we're studying proteins, and their varying structures / roles). I have a lab report to write on it coming up (tomorrow actually), but I want to make sure that I have my science down, before I go writing verbal diarrhea and making a fool of myself. Since I don't have many science friends, this is pretty much my only option (aside from several other forums, but for now I'll start with just the one).

 

My concerns are over what exactly the purpose of the Bradford Reagent is, and why it is used--likewise, why BSA is used / what the purpose of it is. We used the BSA to establish a standard curve, using 0microL (control), 2microL, 4microL, 6microL, and 8microL of the BSA in microtiter plates (done twice to get a comparative average).

 

Then, after establishing a standard curve we took 3microL of 5 different foods (Rice Milk, Coffee Creamer, Soy Sauce, Chili & Sour Cream). We picked these randomly from 20+ different food variants.

 

So to bring us up to speed:

200microL of the Bradford reagent was used within each of the 20 microtiter wells. Well rows A-B contained the 0-8microL of BSA in well #s 1-5. Well rows A-B, #s 6-10 contained the food stuffs. Then the microtiter plate was read by a spectrophotometer at an OD of 595nm.

 

I will provide the data in hopes of getting the best help in analyzing this information, and ensuring I understand the analysis.

 

BSA OD Readout

0microL - 0.00

2microL - .281

4microL - .538

6microL - .748

8microL - .880

 

Food Stuffs OD Readout (3microL each) BEFORE dividing by 3, in order of highest OD readout, to lowest.

1.) Creamer - 1.1505 (0g protein per 2g serving) = 0.00% protein content (5)

2.) Sour Cream - .913 (1g protein per 28.4g serving) = 3.52% protein content (3)

3.) Rice Milk - .189 (.4g protein per 245g serving) = .163% protein content (4)

4.) Chili - .121 (17g protein per 227g serving) = 7.49% protein content (2)

5.) Soy Sauce - .104 (2g protein per 18g serving) = 11.1% protein content (1)

 

(The hyphens are dividers, not minus signs)

 

When looking up the ingredients I was surprised to find that Cremora (the brand of Coffee Creamer used) had no protein content what-so-ever, and while I was expecting Chili to be a close runner for 1st or 2nd highest protein concentration, it was near dead last--this, I am assuming, is due to when the Chili was cooked, the proteins had been denatured? Yet that still doesn't explain why Cremora scored so high (although to get a 1 mg/ml BSA solution ratio we divided the food stuffs results by 3). Which then leads to the next question, what is the difference between denatured proteins and naturally occurring proteins? (Not a Lab question, but just a personal one) For example: why is cooked chicken good for promoting muscle building, as opposed to it raw (aside from obvious food-borne illnesses), but also, why wouldn't it (denatured proteins) be useful for this assay?

 

When looking at the data as a whole, it almost goes perfectly inverse in relationship to what common sense--given the protein contents imply--as far as readouts are concerned.

 

Thanks for reading, I hope I was concise enough in what I am asking! Feel free to leave any feedback, commentary, etc.

[CharonY]

The way Bradford works, denaturation should not influence it by too much. There are a variety of caveats, though. First is the influence of other stuff in your sample. Assuming that you did not include purification step, other compounds that are not proteins may influence the assay. Some may, for instance absorb at 595, whereas other (e.g. high salt levels) may inhibit or promote formation of dye-protein complexes. It is also relevant to note that the efficiency of the dye-complex formation is also dependent on the protein itself. For instance, at the same concentration, BSA generally yields higher ODs than IgG.

 

That being said, there are also some issues with your data. For instance the OD readings are well above the dynamic range of your standard curve or are in the lower range. This is not very helpful for an accurate determination.

 

Overall I would say that the influence of other compounds, especially particulate formation in your sample are playing a bigger role. For proper protein content determination it is generally a prerequisite to have at least one extraction step prior to actual measurement.

[ishbar]

The way Bradford works, denaturation should not influence it by too much. There are a variety of caveats, though. First is the influence of other stuff in your sample. Assuming that you did not include purification step, other compounds that are not proteins may influence the assay. Some may, for instance absorb at 595, whereas other (e.g. high salt levels) may inhibit or promote formation of dye-protein complexes. It is also relevant to note that the efficiency of the dye-complex formation is also dependent on the protein itself. For instance, at the same concentration, BSA generally yields higher ODs than IgG.

 

That being said, there are also some issues with your data. For instance the OD readings are well above the dynamic range of your standard curve or are in the lower range. This is not very helpful for an accurate determination.

 

Overall I would say that the influence of other compounds, especially particulate formation in your sample are playing a bigger role. For proper protein content determination it is generally a prerequisite to have at least one extraction step prior to actual measurement.

 

Those initial yields are to be divided by three (of the foodstuffs), so the 1.1505 comes down to a more reasonable .384, it just becomes problematic for the much smaller numbers, since it makes it difficult to place them relative to the standard curve. Likewise, regardless of numeric value, despite the BSA standard not having as high of values, is it wrong to assume that the line would continue to trend, given the four points + control? i.e., there is enough BSA data to determine a generalized path that the values will trend, and thus accommodate for the higher data values?

 

As far as the Chili / Soy Sauce go, in the Chili sample we had a homogenous mixture. When pipetting 3microL, we took from the top--an orangish slurry (i'm assuming water and lipids)--at the bottom, the "meat and potatoes" of what most expect from chili: beef, onion particulate, and whatever else (beans?). So this may have factored? (I asked the instructor, to which they replied where I got the sample factored very little into it, but the denaturing did...so you telling me otherwise now has me very confused.)

 

I read about the Bradford reagent a little earlier and I saw that acidity can factor into how well the OD reading is. The Cremora carbohydrate percentage is 1g for every 2g serving. So is it safe to assume that--if anything--this might be a key reason as to why the reading may have shown the OD reading as a high protein content? It also has *some* fat, but not nearly as much fat as in SC or the carbs.

[CharonY]

With regard to the dynamic range: the calibration curve is based on the actual output of your measurement system (in this case the OD readout of the photometer). Moreover, for most photometers an OD of over 1 is highly inaccurate and requires dilution before the measurement. PH is certainly a factor. A slurry is not a good thing as particulate matter absorbs or deflects your beam. I.e. for a decent measurement you analyte has to be completely dissolved.

With regard to background: I recall that Biorad had a nice summary of a number of things. You may want to check their website.

 

With regards to denaturation: in the Bradford reagent phosphoric acid is actually used to denature the proteins in order to enhance the complex formation. Also to think about it in a practical way (at least for those familiar with protein analyses). Bradford is based on coomassie. Where else is it used and are the proteins still native?

 

What was meant is possibly that without homogenization the denatured proteins (i.e. cooked meat) will simply accumulate as precipitants. Relatively little of it will be found in the supernatant. I am surprised, though. If you really want to figure out the protein content the first step would be to homogenize it and then get rid of interfering substances.

Edited October 11, 2011 by CharonY

[ishbar]

With regard to the dynamic range: the calibration curve is based on the actual output of your measurement system (in this case the OD readout of the photometer). Moreover, for most photometers an OD of over 1 is highly inaccurate and requires dilution before the measurement. PH is certainly a factor. A slurry is not a good thing as particulate matter absorbs or deflects your beam. I.e. for a decent measurement you analyte has to be completely dissolved.

With regard to background: I recall that Biorad had a nice summary of a number of things. You may want to check their website.

 

With regards to denaturation: in the Bradford reagent phosphoric acid is actually used to denature the proteins in order to enhance the complex formation. Also to think about it in a practical way (at least for those familiar with protein analyses). Bradford is based on coomassie. Where else is it used and are the proteins still native?

 

What was meant is possibly that without homogenization the denatured proteins (i.e. cooked meat) will simply accumulate as precipitants. Relatively little of it will be found in the supernatant. I am surprised, though. If you really want to figure out the protein content the first step would be to homogenize it and then get rid of interfering substances.

 

Thank you, the Bio-Rad site is pretty cool, and while I was only able to glean over it for a moment, it definitely has been bookmarked for future reference! As far as better methods--in pursuit of succinct protein content values--the materials / unknowns were selected by my Lab Professor, it was actually Chili she had the night before, hah! I definitely feel a little more reassured, next we're doing a Western Blot, so it will be exciting to see how that turns out!