Aim:
To estimate the amount of protein in the given sample by Lowry’s method.
Principle:
The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin- Ciocalteay phosphomolybdic phosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acids [Dunn, 13].
The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10 - 10.5. The Lowry method is sensitive to low concentrations of protein. Dunn [1992] suggests concentrations ranging from 0.10 - 2 mg of protein per ml while Price [1996] suggests concentrations of 0.005 - 0.10 mg of protein per ml. The major disadvantage of the Lowry method is the narrow pH range within which it is accurate. However, we will be using very small volumes of sample, which will have little or no effect on pH of the reactionmixture. A variety of compounds will interfere with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents [Dunn, 1992]. Price [1996] notes that ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These substances should be removed or diluted before running Lowry assays.
The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10 - 10.5. The Lowry method is sensitive to low concentrations of protein. Dunn [1992] suggests concentrations ranging from 0.10 - 2 mg of protein per ml while Price [1996] suggests concentrations of 0.005 - 0.10 mg of protein per ml. The major disadvantage of the Lowry method is the narrow pH range within which it is accurate. However, we will be using very small volumes of sample, which will have little or no effect on pH of the reactionmixture. A variety of compounds will interfere with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents [Dunn, 1992]. Price [1996] notes that ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These substances should be removed or diluted before running Lowry assays.
Reagents:
A. 2% Na2CO3 in 0.1 N NaOH
B. 1% NaK Tartrate in H2O
C. 0.5% CuSO4.5 H2O in H2O
D. Reagent I: 48 ml of A, 1 ml of B, 1 ml C
E. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water
F. BSA Standard - 1 mg/ ml
Procedure:
• 0.2 ml of BSA working standard in 5 test tubes and make up to 1ml using distilled water.
• The test tube with 1 ml distilled water serve as blank.
• Add 4.5 ml of Reagent I and incubate for 10 minutes.
• After incubation add 0.5 ml of reagent II and incubate for 30 minutes
• Measure the absorbance at 660 nm and plot the standard graph .
• Estimate the amount of protein present in the given sample from the standard graph.
Tabulation:
|
Sample
|
Volume
of BSA
(ml)
|
Volume
of Distilled water (ml)
|
Concentration
of BSA (mg/ml)
|
Volume
of alkaline copper sulphate (ml)
|
Incubation
for 10 minutes
|
Volume
of Folin Reagent (ml)
|
Incubation
for 20 minutes in dark
|
Absorbance
at 660nm
|
|
Blank
|
|
|
|
|
|
|
||
|
S1
|
|
|
|
|
|
|
||
|
S2
|
|
|
|
|
|
|
||
|
S3
|
|
|
|
|
|
|
||
|
S4
|
|
|
|
|
|
|
||
|
S5
|
|
|
|
|
|
|
||
|
Unknown
|
|
|
|
|
|
|
Notes:
1. If the sample is available as a precipitate, then dissolve the precipitate in 2N NaOH.
2. Peterson has described a precipitation step that allows the separation of the protein sample from interfering substances and also consequently concentrates the protein sample, allowing the determination of proteins in dilute solution. Peterson's precipitation step is as follows:
a. Add 0.1 mL of 0.15% deoxycholate to 1.0 mL of protein sample.
b. Vortex, and stand at room temperature for 10 min.
c. Add 0.1 mL of 72% TCA, vortex, and centrifuge at 10000 rpm for 30 min.
d. Decant the supernatant and then dissolve the pellet in 2N NaOH.
3. The reaction is very pH-dependent, and it is therefore important to maintain the pH between 10 and 10.5. Take care, therefore, when analyzing samples that are in strong buffer outside this range.
4. The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance.
5. The Vortex step is critical for obtaining reproducible results. The Folin reagent is only reactive for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing.
6. The assay is not linear at higher concentrations. Ensure, therefore, that you are analyzing your sample on the linear portion of the calibration curve.
7. A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended.
8. One disadvantage of the Lowry method is the fact that a range of substances interfere with this assay, including buffers, drugs, nucleic acids, and sugars. In many cases, the effects of these agents can be minimized by diluting them out, assuming that the protein concentration is sufficiently high to still be detected after dilution. When interfering compounds are involved, it is, of course, important to run an appropriate blank. Interference caused by detergents, sucrose, and EDTA can be eliminated by the addition of SDS. The best alternative in this case is to do Lowry-TCA or Peterson.
9. Modifications to this basic assay have been reported that increase the sensitivity of the reaction. If the Folin reagent is added in two portions, vortexing between each addition, a 20% increase in sensitivity is achieved. The addition of dithiothreitol 3 min after the addition of the Folin reagent increases the sensitivity by 50%.
10. The amount of color produced in this assay by any given protein (or mixture of proteins) is dependent on the amino acid composition of the protein(s) (see Introduction). Therefore, two different proteins, each for example at concentrations of 1 mg/mL, can give different color yields in this assay. It must be appreciated, therefore, that using BSA (or any other protein for that matter) as a standard only gives an approximate measure of the protein concentration. The only time when this method gives an absolute value for protein concentration is when the protein being analyzed is also used to construct the standard curve. The most accurate way to determine the concentration of any protein solution is amino acid analysis.
Interfering Reagents for Lowry
Many detergents, Urea, Guanidine HCl, high sucrose, Ammonium sulphate, >0.1M TrisHCl, >1MNa Acetate or Na Phosphate, EDTA, reducing agents, etc.
2. Peterson has described a precipitation step that allows the separation of the protein sample from interfering substances and also consequently concentrates the protein sample, allowing the determination of proteins in dilute solution. Peterson's precipitation step is as follows:
a. Add 0.1 mL of 0.15% deoxycholate to 1.0 mL of protein sample.
b. Vortex, and stand at room temperature for 10 min.
c. Add 0.1 mL of 72% TCA, vortex, and centrifuge at 10000 rpm for 30 min.
d. Decant the supernatant and then dissolve the pellet in 2N NaOH.
3. The reaction is very pH-dependent, and it is therefore important to maintain the pH between 10 and 10.5. Take care, therefore, when analyzing samples that are in strong buffer outside this range.
4. The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance.
5. The Vortex step is critical for obtaining reproducible results. The Folin reagent is only reactive for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing.
6. The assay is not linear at higher concentrations. Ensure, therefore, that you are analyzing your sample on the linear portion of the calibration curve.
7. A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended.
8. One disadvantage of the Lowry method is the fact that a range of substances interfere with this assay, including buffers, drugs, nucleic acids, and sugars. In many cases, the effects of these agents can be minimized by diluting them out, assuming that the protein concentration is sufficiently high to still be detected after dilution. When interfering compounds are involved, it is, of course, important to run an appropriate blank. Interference caused by detergents, sucrose, and EDTA can be eliminated by the addition of SDS. The best alternative in this case is to do Lowry-TCA or Peterson.
9. Modifications to this basic assay have been reported that increase the sensitivity of the reaction. If the Folin reagent is added in two portions, vortexing between each addition, a 20% increase in sensitivity is achieved. The addition of dithiothreitol 3 min after the addition of the Folin reagent increases the sensitivity by 50%.
10. The amount of color produced in this assay by any given protein (or mixture of proteins) is dependent on the amino acid composition of the protein(s) (see Introduction). Therefore, two different proteins, each for example at concentrations of 1 mg/mL, can give different color yields in this assay. It must be appreciated, therefore, that using BSA (or any other protein for that matter) as a standard only gives an approximate measure of the protein concentration. The only time when this method gives an absolute value for protein concentration is when the protein being analyzed is also used to construct the standard curve. The most accurate way to determine the concentration of any protein solution is amino acid analysis.
Interfering Reagents for Lowry
Many detergents, Urea, Guanidine HCl, high sucrose, Ammonium sulphate, >0.1M TrisHCl, >1MNa Acetate or Na Phosphate, EDTA, reducing agents, etc.
0 comments:
Post a Comment