Bradford Test for Protein

(adapted from Kirk Brown; Tracy High School)

Introduction

You have seen how to determine the presence or absence of various organic molecules with specific testing reagents such as Benedicts (tests for reducing sugars) or Lugol’s Iodine (tests for starch) or even Biuret (tests for proteins).  These reagents were qualitative in nature.  They gave a color change but that was only used to determine if they were present or absent.  There are methods that can be used to determine their concentration.  Qualitative measures determine if they are there or not, by color or some other quality.  Quantitative measures determine the amount or quantity present.

Quantitative estimation of the total protein content of a sample is frequently necessary in cell physiological and biochemical studies. Several methods of determining the total protein content of a sample have been developed and widely used during this century. One of the simplest and most sensitive is the "Bradford" assay, which was introduced in the mid-1970s. This assay is based on the binding specificity of the dye Coomassie Brilliant Blue-G250 for protein molecules but not for other cellular constituents. This organic dye binds specifically to tyrosine side chains.

The binding of the dye to protein shifts the peak absorbance of the dye. Unbound Coomassie Blue absorbs light maximally at a wavelength of 465 nm, while the absorption maximum is at 595 nm when the dye is bound to protein. The absorbance of light by the dye-protein complex at 595 nm is proportional to the amount of protein bound (over a limited range); i.e. there is a linear relationship between absorbance and the total protein concentration of the sample over a narrow range.(source: http://www.acad.carleton.edu/curricular/BIOL/resources/rlink/lab1p4.html)

So the main idea is to have a series of proteins that you know the concentration of protein and mix them with Bradford reagent.  Once you determine the absorbance with a series of known proteins, you can graph protein concentration vs. absorbance.  By mixing unknown proteins with the same reagent, you can use the graph to work backwards from absorbance and determine protein concentration (see Figure 1).

Procedure
Note: do all determinations in duplicate by working in collaboration with another group.

Construction of Standard Curve  (note: some browsers change microliters  and micrograms (µg/µl) to ml and mg. Double check with teacher.

  1. To determine the concentration of protein in an unknown solution it is first necessary to make a control standard (done by teacher) and create a standard curve using a series of known protein concentrations.  We will be using BSA or Bovine Serum Albumin (cow blood protein).  The stock solution is a 1 mg/ml solution.

  2. You will be given a series of dilute solutions.  These solutions were made using the formula C1V1=C2V2 (C= concentration; V= volume) to make 100 µl of each of the following BSA dilutions. 0.2, 0.4, 0.6, 0.8 and 1.0  mg/ml (µg/µl).  A 1X strength Phosphate Buffered Saline (PBS) was used to do the dilutions.

  3. Milk dilutions of 1:10, 1:50, 1:100 will be provided and were made with 1X PBS.

What you will do:

  1. First of all: NEVER TOUCH ANY PART OF THE CUVETTE OTHER THAN THE VERY, VERY, VERY TIPPITY TOP. Also make sure you use a kimwipe to clean the cuvette before inserting into the Spec 20.

  2. Label the tippity top of the cuvettes… 0.2, 0.4, 0.6, 0.8, and 1.0 BSA, Blank (control), Milk (which ever type you are assigned) dilutions of 1:10, 1:50 AND 1:100.

  3. Add 50 µl of each of the different stock 5 BSA solutions into the appropriately labeled cuvettes and 50 µl of the milk dilutions (total 8 cuvettes); add 50µl of 1X PBS to the Blank (the 9th cuvette).

  4. Add 2.5 ml (milliliter) of Bradford Reagent to each cuvette.  Mix well by shaking gently from the tippity top and let sit 5 minutes.

  5. Make sure the Spectrophotometers have been set to 595nm and let warm up for a min of 15 minutes. Zero the machine as shown by the instructor (no tube used) then set Transmittance to 100% using the Blank cuvette (tube # 9) that has 50 ml of 1X PBS and 2.5ml of Bradford reagent.

  6. Remove Blank tube and place your 0.2mg/ml tube in the spectrophotometer.  Make sure you record absorbance (not transmission) at 595 nm.

  7. You should check that the machine isn’t drifting from the Blank.

  8. Repeat for the rest of the cuvettes (including the milk).


Why Do Dilutions







 





Example: In microbiology labs scientists perform a three step 1:100 serial dilution of a bacterial culture (see figure below). The initial step combines 1 unit volume culture (10 ul) with 99 unit volumes of broth (990 ul) = 1:100 dilution. In the next step, one unit volume of the 1:100 dilution is combined with 99 unit volumes of broth now yielding a total dilution of 1:100x100 = 1:10,000 dilution. Repeated again (the third step) the total dilution would be 1:100x10,000 = 1:1,000,000 total dilution. The concentration of bacteria is now one million times less than in the original sample.  We are using the same technique to dilute milk from the carton, up to a 1:100 dilution



Determination of Unknown Protein Concentration in Milk.

Graph the absorbance vs BSA protein concentration.  Remember the dependent variable goes on the Y and the independent goes on the X axis.

Draw line of best fit through your points. Using the trendline also check off (under options) that you want the equation of the line. You will use the equation to solve for x (unknown protein) as well as hand plot on your graph.

Plot your unknown absorbance on the graph and use graph to estimate protein concentration in milk.  Use the dilution factors to actually get back to the quantity of protein in a serving of milk.

Using the given protein content (from the milk carton) calculate the % error between your observed values vs. the expected results (milk carton).  The formula for % error is

(Observed – Expected / Expected) x 100

 

Figure: 1

Sample Graph and use to determine protein concentration after getting absorbance of unknown.



Helpful Hints for Calculating Amount of Protein in One Serving of Milk:

Remember: 1000 µl = 1 ml; 1000 mL = 1 L;  1000µg = 1 mg; 1000 mg = 1 g

So; if you have 0.396 µg/µl then that is equivalent to 0.396 mg/ml, etc…

You will need to figure out how many micrograms/microliter of protein are in milk and compare to your values.



 Rubric:

Rubric for Got Protein:  (23 points)

Title, Data Tables, Standard Curve Graph, Calculations including % error,  Summary/ Conclusion; References (2 min).

Title: must be descriptive and include the IV and DV of your lab experiment. (1 point)

Results:

Using class averages (if available) generate a Standard Curve for BSA (includes descriptive title, labeled axis, trendline and equation (y=mx+b).  (4 points)

Calculations:  (please do hand calculations, very difficult to type)

a) Given (expected); example the information on the side of the milk carton reads 9g/240 mL; convert to micrograms/microliter. Soy milk (6g/ 240 mL), Rice milk (1g/240mL), Almond milk 1g/240 mL (2 points)

b) Using the absorbance of your 3 unknown milk dilutions (0.1, 0.01, and 0.001)... solve for "x" using the equation from the BSA standard curve. Remember your values will be in micrograms/microliter... multiply each calculation by either 10 (for the 0.1 dilution); 100 (for the 0.01 dilution), 1000 (for the .001) to get the actual value of your unknowns (6 points)

c) Using the given protein content (from the milk carton) calculate the % error between your observed values vs. the expected results (milk carton) for each dilution.  (3 points)

The formula for % error is

(Observed – Expected / Expected) x 100

Summary/ Conclusion  (5 points)

Discuss the purpose and results of the experiment, the use of a spectrophotometer to determine a standard curve to find an unknown concentration of a substance. Discuss the possible places where errors could occur during the set-up and execution of the measurement of absorbance. Don't forget to include the use of a blank. Do not forget to include citations after sentences for information you used from other sources. Avoid pronouns.

References: (2 points)

Use correct format APA (http://www.bibme.org) or Son of citation