Experiment 1:  Protein determination via the Bradford method

Literature:

Bradford.  Analytical Biochemistry. 72: 248-254. (1976)
Peterson.  Methods in Enzymology. 91: 95-119. (1983)
Compton & Jones.  Analytical Biochemistry. 151: 369-374. (1985)

Theory

     This method is based on the idea that an acid solution of Coomassie Brilliant Blue G can be changed from 465nm too 595nm via protein binding. The resulting complex can be measured at 578nm in a filter photometer, so that the protein content of a solution after preparation can be used to create a calibration curve. The method was first described by Marion Bradford in 1976 and this is why it is so named. The method uses only one reagent and is quick and sensitive; it is also insensitive to buffers, reduction agents, and complex BILDNER, but is degenerated by bases and detergents. Numbers variations have been developed in order to increase the sensitivity and/or decrease errors caused by interferents. We will be applying reagents made by and a procedure developed by the Roth company.

     Coomassie Brilliant Blue (CBB) is a tri-anilo-methane wool dye discovered at the end of the 19th century and is named after the West African city Kumasi (Coomassie in English; it is now in Ghana). It binds to bases (arginine most of all) and aromatic at a rate of approx. 1mg CBB per 1mg prtein. CBB-G250 is greenish with an anionic absorption maximum of 575-585nm and is used in the dection of proteins in experiments 1, 6, 8, and 12. Without aryl-methyl groups, CBB receives the additive R250 (reddish, with a maximum of 555-570nm) and is rarely used.


Figure 1. Structural formula of CBB-G250.
Cation Neutral Anion
470nm 650nm 595nm
red green blue
pH 0.30            pH 1.25

Objective

     The protein contents of an unknown ß-gal-ase solution and an unknown bovine serum albumin (BSA) solution will be assesed using calibration curve created with a known protein concentration of bovine serum albumin.

Procedure:

You should have the following solutions at your disposal:

  • BSA stock soltuion: 1mg BSA / 1mL water
  • ß-gal-ase soltuion, with a dilution of 1:1500
  • BSA solution of unknown concentration
  • Coomassie reagent (= Bradford reagent, here Roti-Quant 5x from the Roth company)

     After creating the calibration curve, measure the following BSA dilutions into a test tube, using a graduated cylinder.

Table 1. Composition of the required reagents.
BSA Solution # µg BSA/mL Stock Solution (mL) mL H2Odist Vf (mL)
S 20 0.4 (stock soln) 19.6 20.0
1 15 7.5 (soln S) 2.5 10.0
2 12.5 6.25 (soln S) 3.75 10.0
3 10 5.0 (soln S) 3.75 5.0
4 7.5 5.0 (soln 1) 10.0 10.0
5 5 5.0 (soln 3) 5.0 10.0
6 2.5 5.0 (soln 5) 5.0 10.0

NOTE:  The following steps involves contact with reagents containing H3PO4; be sure your safety goggles are securely fastened!

     Into 7 different disposable half-micro (?) cuvettes, place 1 mL of each newly-made solution along with 1mL of the known ß-gal-ase solution and 250µL of the Roti-Quant 5x solution. The cuvettes should be labelled with a felt-tipped marker.

     The measurements of the unknown BSA solution serves as an exercise in pipetting small quantities with an automatic pipette. These volumes should be taken 5x and the variaces recorded. Pay close attention to the following points, as they may throw off your experimental results: quality of the pipette, immersion depth of the tip, presence of droplets on the outside of the tip, presence of air bubbles, precipitate clumping in solution or on the walls of the reaction vesicle. Immerse your pipette tip in the unknown BSA solution and take up 1 µL. Deposit it in the cuvette (already containing the water), close with parafilm, and swirl, before adding the required amount of the Roti-Quant reagent and swirling again in the same manner.

Table 2. BSA stock solution and ß-gal-ase requirements for preparation of the calibration curve.
mprotein (µg) VBSA VRoti-Quant 5x (µL)
15 1mL solution 1 250
12.5 1mL solution 2 250
10 1mL solution 3 250
7.5 1mL solution 4 250
5 1mL solution 5 250
2.5 1mL solution 6 250
0 1mL H2O 250
? 1mL ß-gal-ase dilution 250
? 1µL unkown + 1mL H2O
(see following text)		 250

     All of the above preparations should be left to stand for 5 minutes before being measured into a 578nm filter for the Eppendorf photometer. The cuvettes must be used in such a manner that the thickness of the optical path must be 5mm, because of the high optical absorption of the container itself. The extinction should take place slowly over the next half hour; if the protein coagulates, no more useful measurements are possible.

     Use the resulting values to create a caibration curve and characterize the concetration of the remaining ß-gal-ase; this will be used in a 1500x dilution in kinetics experiments 2 and 3 and thus, this experiment should be completed within the course of a single day. Hand in all 5 measured values, along with the average, standard deviation, and a discussion of sources of error in your lab report.

[diagram of a sample calibration curve for the Roti-Quant 5x method]


Questions to be addressed in your report, separate from the discussion:
Refer to Appendix A

  1. How can protein determination methods be quality-controlled?
  2. Which are the most sensitive and the most insensitive protein determination methods?
  3. Which protein determination method are senstitive to reduction agents, and which to complexing agents?
  4. Which amino acids can be oxidized and which protein determination method should be used?
  5. What is the difference between Coomassie Brilliant Blue G and R?
  6. What is the difference between the red and the blue forms of Coomassie Brilliant Blue G in terms of pH?
  7. What is the control protein determination method in the Bradford Method, or how would the concentration of the BSA stock solution be checked/calibrated?
  8. Is the calibration curve in the Bradford method straight or crooked/curved? What is the significance of the shape?
  9. In what sensitivity range does the Bradford method work?
  10. Which amino acids bind the CBB pigments?
  11. How can a protein be determined without the help of its amino acid composition?
  12. Draw the spectrum of a colourless protein and of DNA. At what wavelength do peptide bonds and aromatic amino acids absorb?
  13. How can a protein be determined in the presence of DNA?
  14. What units are used to describe protein characterization?
  15. Should a measurement of protein concentration be taken from the unknown solution or from a dilution thereof?
  16. Does the volume of the original solutions play a roll in the determination of the concentrations?
  17. What influences do the thickness of the cuvette and the wavelength of the photometer have on the recorded measurements?
  18. Does the molecular mass of the protein play a roll in its detection?
  19. What errors are associated with pipetting small volumes (0.5-2µL)?

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Reference A

Protein determination methods

Method Principle Max. Sensitivity Pros Cons
weighing   100 µg simple high water content
Kjeldahl requires concentrated H3PO4 + metal catalysts, titration of free NH3   little amino acid dependence tedious
ASA total hydrolysis, determination of all free amino acids Ninhydrin: 10 µg
OPA: 10 ng		 simple, amino acid independent tedious
Biuret A545 of Cu2+-N4 complexes 100 µg simple, little amino acid dependence low sensitivity, amine (eg. Tris) interference
Lowry-Folin reduction of Mo6+/W6+ via Cu1+-N4 complexes and oxidizable aromatic amino acids; A720 5 µg simple, sensitive non-linear results, lots of interference
BCA bicinchonic acid complexation; Cu1+ => A562 5 µg sensitive with less intereference picked up than in Lowry-Folin  
UV
(Gill + von Hippel)		 E = nTrp*5690 + nTyr*1280 + Cys*120 50 µg quick, non-destructive influenced by aromatics
UV
(Scopes)		 mg/mL = A205/(27+120+*[A280/A205]) 5 µg sensitive, amino acid-independent lots of disturbance around
l = 205 nm
UV
(Warburg + Christian)		 mg/mL = 1.55*A280 - 0.76*A260 50 µg independent of nucleic acid presence  
UV
(Groves)		 isoabsorption A224 - A236 5 µg sensitive, independent of nucleic acid presence  
Bradford basic and aromatic amino acids bind to Coomassie Brilliant Blue; 465nm > 595nm 1 µg quick (5 min) detergents and alkali ions interfere; amino acid-independent
Turbidimetry protein + TCA => suspension => A570 5 µg quick instable
PAGE colors of electrophoretically separated protein bands 1 µg very sensitive tedious
Special methods:  Proof of absorption by a prosthetic group, enzyme activity test, immunological proof

Back to experiment 1 questions


Things to know:

  • biuret
  • Lowry-Folin
  • Coomassie Brillian Blue
  • bicinchoninic acid
  • turbimetry
  • UV spectroscopy with and without DNA
  • total hydrolysis and/wrt amino acid analysis
  • protein-specific methods
  • LIT: Peterson, (1983). Methods in Enzymology, 91, 119.


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