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Proteins Protocol
PROTEINS (LOWRY) PROTOCOL
1. INTRODUCTION
The “Lowry Assay: Protein by Folin Reaction” (Lowry et al. , 1951) has been the most widely used method to estimate the amount of proteins (already in solution or easily-soluble in dilute alkali) in biological samples. First the proteins are pre-treated with copper ion in alkali solution, and then the aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic acid present in the Folin Reagent. The end product of this reaction has a blue color. The amount of proteins in the sample can be estimated via reading the absorbance (at 750 nm) of the end product of the Folin reaction against a standard curve of a selected standard protein solution (in our case; Bovine Serum Albumin-BSA- solution). Please refer to Sections 22.7.5 and 22.7.6 at “ Methods for General and Molecular Bacteriology ” by Gerhardt et al. (1994) for further information and useful tips. We have this book at the Raskin Lab (Loc: 4217).
2. MATERIALS and CHEMICALS
- Clean Glassware
- Glass tubes : of any kind, approx. 10 mL
- Semi-micro cuvettes : Standard type plastic disposable cuvettes with 10 mm path length, sample capacity of 1.5-3 mL (FisherBrand; 14-385-942): Different than the total carbohydrates measurements, we have to use these semi-micro cuvettes, since the volume of the end product we need to measure is only 1.3 mL.
- Spectrophotometer : that goes up to 750nm (Loc: 4215).
- Bovine Serum Albumine : Albumin bovine serum, Cohn fraction V (Sigma-Aldrich; A9647) (Loc: 4217, +4oC small fridge)
- Folin Reagent : Folin and Ciocalteu’s Phenol Reagent, 2N, Sigma (Loc: 4130)
- Sodium tartrat : Na 2 Tartrat.2(H 2 O) (Loc: 4130)
- Copper sulfate : CuSO 4 .5(H 2 O) (Loc: 4130)
- Other chemicals : NaOH, Na 2 CO (^3) 3. PREPARING the SOLUTIONS
- The Lowry solution is a mixture of the above chemicals, except the Folin Reagent. The Lowry solution should be prepared fresh, at the day of measurement. Though the individual solutions for the Lowry solution can be prepared in advance and then mixed at the day of measurement. Personal note: Different than the protocol given by Gerhardt et al. (1994), I dissolve CuSO 4 .5(H 2 O) and Na 2 Tartrate.2(H 2 O) separately. Please compare this protocol and the one given by Gerhardt et al. (1994). You can find my calculations for this comparison at the end of this protocol. Use ddI water.
- Solution A is a dilute alkali solution. 2N Folin and Ciocalteu’s Phenol Reagent contains hydrochloric and phosphoric acids. So, wear gloves, goggles, lab coat, and work carefully
SOLUTION A (alkaline solution) (for 500 mL) 2.8598 g NaOH 14.3084 g Na 2 CO 3
SOLUTION B (for 100 mL) 1.4232 g CuSO 4 .5(H 2 O)
SOLUTION C (for 100 mL) 2.85299 g Na 2 Tartrate.2(H 2 O)
Proteins Protocol
LOWRY SOLUTION (fresh; 0.7 mL/ sample)
Solution A + Solution B + Solution C with a ratio (vol:vol) of 100:1:1.
FOLIN REAGENT (instant fresh; 0.1 mL/ sample)
5 mL of 2N Folin and Ciocalteu’s Phenol Reagent + 6 mL of ddI water
- This solution light-sensitive. So it should be prepared at the last 5 min of the first sample incubation (see the procedure) and kept in an amber container.
- At the protocol given by Gerhardt et al. (1994), the dilution ratio for the Folin and Ciocalteu’s Phenol Reagent is 1:1, resulting in a 1N Folin reagent.
BSA STANDARD PROTEIN SOLUTION (fresh)
Although BSA is a water-soluble protein, it takes time to dissolve it completely. So, prepare this stock solution and keep it mixed i.e., for 1 hour before starting the experiments.
- Weigh 0.05 g of BSA and add to a 500 mL volumetric flask containing ddI water
- Stir well to dissolve and adjust the volume to 500 mL with ddI water: Final concentration of the stock is 100 mg BSA/L.
- Prepare dilutions* in 15 mL Croning tubes, following the recipe at Table 3.1.
Table 3.1 Dilutions from the BSA stock solution (100 mg/L) for the standard curve
V ddI water V of stock BSA solution Final conc.
10 mL 0 mL 0 mg/L 8 mL 2 mL 20 mg/L 6 mL 4 mL 40 mg/L 4 mL 6 mL 60 mg/L 2 mL 8 mL 80 mg/L 0 mL 10 mL 100 mg/L
*POINTS OPEN FOR DISCUSSION : I’ve been preparing standard curves at the above range for my measurements. I do not need to dilute the BLANK and SUPERNATANT samples from the EPS extraction experiments, but I usually need to dilute my DOWEX-extracts (1:5 to 1:10 dilutions). Depending on the general character of the sample and the expected protein values for the supernatant and the DOWEX-extract, a higher-range calibration curve can be prepared to avoid sample dilution. Also at the protocol given by Gerhardt et al. (1994), the concentration of the stock standard solution is given as 500 mg/L, instead of 100 mg/L. Though since we’re preparing the stock solution fresh every time, I think we can continue to prepare a stock solution of 100 mg/L, if we continue to use the above range for the standard curve.
Proteins Protocol
5. CLEAN-UP and WASTE DISPOSAL - When done with the spec. readings, collect all the samples (in the semi-micro cuvettes), left-over Lowry and Folin solutions in containers; label - Collect the emptied semi-micro disposable cuvettes in a plastic bag; label - Collect all the generated waste to a designated area to be picked-up.
- Get a CWM-TRK-01 form (Request for Pickup of Chemical Waste) from the Lab. Manager and fill it out for the liquid and solid wastes to be picked up and disposed. You can find the copies of the forms I filled out either at Shaoying Qi’s office or in the folder I left to Eberhart Morgenroth and Julie Zilles.
- Rinse all the glassware and then clean with soapy water, rinse well again
- Clean the work space well
6. NOTES
- Different proteins give different responses to the Lowry method. So pick a particular commercially available protein as the standard and keep using it for all of your experiments.
- It’s advised to run the samples in duplicates (Gerhardt et al., 1994), though I run them in triplicates.
- Always run blanks. In our case, the blank is the first sample of the standard curve (0 mg BSA/L).
- Please refer to section 22.7.5.b (2nd^ paragraph) at Gerhardt et al. (1994) for the additional treatment of the samples containing proteins which are not soluble in dilute alkali.
- Commonly used detergents, reducing agents, buffers, ammonium sulfate > 0.15%, 1-5M of many salts, some sugars, glycine, Tris buffer, some chelating reagents, compounds with sulfhydryl groups, amino acids, phenol, usually interfere with the Lowry assay.
7. REFERENCES
Gerhardt, P.; Murray, R.G.E.; Wood, W.A.; Krieg, N.R. (1994) “ Methods for General and Molecular Bacteriology ”, ASM, Washington DC, ISBN 1-55581-048-9, p 518.
Frolund, B.; Palmgren, R.; Keiding, K.; Nielsen, P.H. (1996). “Extraction of extracellular polymers from activated sludge using a cation exchange resin”, Water Res 30 (8), pp. 1749-1758.
Lowry, O.H.; Rosenbrough, N.J.; Farr, A.L.; Randall, R.J. (1951) “Protein measurement with the Folin Phenol Reagent”, J Biol Chem 193 , pp. 265-275.
Proteins Protocol
8. CALCULATIONS
The recipes for the solutions given by Gerhardt et al. (1994) and the ones presented in this protocol seem to be different. Though, when the amounts of chemicals in the final reaction volume are calculated, it’s clear that the differences are not significant.
Gerhardt et al. (1994) This protocol
Reagent A Solution A
4 g in 1 L 0.1 N NaOH 2.8598 g in 500 mL 0.143 N
20 g in 1 L 0.1 N Na 2 CO 3 14.3084 g in 500 mL 0.135 N
Reagent B Solution B
0.5 g in 100 mL 0.02 M CuSO 4 .5(H 2 O) 1.4232 g in 100 mL 0.057 M
1% (w:v) 0.052 M Na 2 Tartrate -- --
Solution C
Na 2 Tartrate.2(H 2 O) 2.85299 g in 100 mL 0.124 M
Reagent C Lowry Solution
50 mL ReagA + 1 mL ReagB 100+1+1 mL SolA+SolB+SolC
0.1N 0.1N 0.0004M 0.001M
NaOH Na 2 CO 3 CuSO 4 .5(H 2 O) Na 2 Tartrate.2(H 2 O)
0.14N
0.132N
0.00056M
0.00121M
Reagent D Folin Solution
1:1 dilution 1 N 5:11 dilution 0.83 N
Reaction recipe:
0.2 mL sample +1 mL ReagentC
0.5 mL sample + 0.7 mL Lowry Sol. + 0.1 mL Folin Sol. = 1.3 mL
Amounts in the final reaction volume of 1.3 mL
0.077 N 0.077 N 0.000308 M 0.00077 M 0.077 N
NaOH Na 2 CO 3 CuSO 4 .5(H 2 O) Na 2 Tartrate.2(H 2 O) Folin C.Phenol Reag.
0.074 N
0.071 N
0.000302 M
0.00065 M
0.064 N
