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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 ...
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Proteins Protocol
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
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
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.
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.
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
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)
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.