ml/cl protein assays - g-biosciences · g-biosciences’ ml & cl protein assays are two...
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G-Biosciences 1-800-628-7730 1-314-991-6034 [email protected]
A Geno Technology, Inc. (USA) brand name
think proteins! think G-Biosciences www.GBiosciences.com
466PR-01
ML/CL Protein Assays Modified and Compatible Protein Assays
based on Lowry’s Methods
(Cat. # 786-1075, 786-1076, 786-1077, 786-1078,
786-1082, 786-1083, 786-1084, 786-1085)
Page 2 of 13
INTRODUCTION ................................................................................................................. 3
ITEM(S) SUPPLIED .............................................................................................................. 3
ML PROTEIN ASSAY ........................................................................................................ 3
CL PROTEIN ASSAY ......................................................................................................... 4
STORAGE CONDITIONS ...................................................................................................... 4
REAGENT COMPATIBILITY .................................................................................................. 4
REPONSE TO VARIOUS PROTEINS: ..................................................................................... 6
ML PROTEIN ASSAY PROTOCOLS ....................................................................................... 6
STANDARD ML-PROTEIN ASSAY PROTOCOL (5ML ASSAY TUBES) ................................. 6
PREPARATION BEFORE USE: ...................................................................................... 6
ML PROTEIN ASSAY .................................................................................................... 7
MICROTUBE ML-PROTEIN ASSAY PROTOCOL (1.5-2.0ML ASSAY TUBES) ...................... 7
PREPARATION BEFORE ASSAY ................................................................................... 7
ML PROTEIN ASSAY .................................................................................................... 7
MICROTITER PLATE ML-PROTEIN ASSAY PROTOCOL (96 WELL PLATE) ......................... 8
PREPARATION BEFORE ASSAY ................................................................................... 8
ML PROTEIN ASSAY .................................................................................................... 8
CL (COMPATIBLE LOWRY) PROTEIN ASSAY PROTOCOLS ................................................... 9
STANDARD CL-PROTEIN ASSAY PROTOCOL (5ML ASSAY TUBES) ................................... 9
PREPARATION BEFORE USE: ...................................................................................... 9
CL PROTEIN ASSAY ..................................................................................................... 9
MICROTUBE CL-PROTEIN ASSAY PROTOCOL (1.5-2.0ML ASSAY TUBES)...................... 10
PREPARATION BEFORE USE: .................................................................................... 10
CL PROTEIN ASSAY ................................................................................................... 10
TROUBLESHOOTING SECTION.......................................................................................... 11
REFERENCES .................................................................................................................... 12
RELATED PRODUCTS ........................................................................................................ 12
Page 3 of 13
INTRODUCTION
G-Biosciences’ ML & CL Protein Assays are two separate variations of protein assay
based on widely cited protein assay by Lowry et. al. (1951). The ML Protein Assay is a
modified Lowry version that is compatible with a wide variety of detergents used in
protein research. The CL Protein Assay, on the other hand, is reducing agent compatible
version, suitable for protein samples contain reducing agents (such as dithiothreitol
(DTT), ß-mercaptoethanol and TCEP) and a wide variety of commonly used laboratory
agents that are known to interference with Lowry’s protein assays (Table 1). The CL
Protein Assay uses G-Biosciences’ proprietary Universal Protein Precipitating Agent
(UPPA™
). The UPPA agent cleans the protein samples from interfering agents prior to
performing colorimetric reaction.
ITEM(S) SUPPLIED
ML Protein Assay
Description
Cat# 786-
1075
Cat. # 786-
1076
Cat. # 786-
1082
Cat. # 786-
1083
Folin’s Reagent 4x250ml 4x250ml 4x250ml 4x250ml
Copper Solution 125ml 125ml 125ml 125ml
Reagent D 2.5ml 2.5ml 2.5ml 2.5ml
BSA Protein Standard
[2mg/ml] 5ml - - -
Non Animal Protein
Standard [2mg/ml] - 5ml - -
Pre-Diluted BSA Protein
[0.1-1mg/ml] - - 6 x 5ml -
Bovine ɣ Globulin
Standard [2mg/ml] - - - 5ml
The kit components are suitable for 5,000 micro-well assays, 1,000 microtube (1.5-2ml)
assays and 250 Standard (5ml tube) assays.
Page 4 of 13
CL Protein Assay
Description
Cat# 786-
1077
Cat. # 786-
1078
Cat. # 786-
1084
Cat. # 786-
1085
Folin’s Reagent 4x250ml 4x250ml 4x250ml 4x250ml
Copper Solution 125ml 125ml 125ml 125ml
Reagent D 2.5ml 2.5ml 2.5ml 2.5ml
UPPATM
I 125ml 125ml 125ml 125ml
UPPATM
II 125ml 125ml 125ml 125ml
BSA Protein Standard
[2mg/ml] 5ml - - -
Non Animal Protein
Standard [2mg/ml] - 5ml - -
Pre-Diluted BSA Protein
[0.1-1mg/ml] - - 6 x 5ml -
Bovine ɣ Globulin
Standard [2mg/ml] - - - 5ml
The kit components are suitable for 1,000 microtube (1.5-2ml) assays and 250 Standard
(5ml tube) assays.
STORAGE CONDITIONS
The kit is shipped at ambient temperature. Upon arrival, store UPPA™-I and UPPA™-II at
room temperature away from direct sunlight. The remaining kit components should be
stored in dark and refrigerated in its original box. When stored properly, the kit is stable
for 1 year.
REAGENT COMPATIBILITY
Table-1: A selection of compounds and their maximum concentrations compatible with
the ML and CL Protein Assay.
Reagent ML Protein Assay CL Protein Assay
Amino Acids Compatible -
Ammonium Sulfate 0.5M 40%
β-Mercaptoethanol X 5%, 15%*
Brij® 35 1% 1%
Calcium Chloride 0.05M -
CHAPS 1% 4%
CHAPSO 1% 1%
CTAB - 1M
Deoxycholate 1% -
Digitonin 0.3% -
DTT 0.001M 0.1M, 0.35M*
EDTA 0.025M 0.1M
Page 5 of 13
Reagent ML Protein Assay CL Protein Assay
Glycerol - 30%
Guanidine.HCl 0.4M 6M
Guanidine Thiocynate - 6M
HEPES - 0.1M
Hydrochloric Acid 0.5M -
Imidazole - 0.5M
Iodoacetamide - 15mM
Laemmli Buffer (w/5% β-Mercaptoethanol) - Compatible
NP-40 2% -
Octaethyleneglycol dodecyl ether 0.2% -
Octyl Glucoside 1% -
Phosphate Buffer - 0.2M
Sarcosyl - 1%
SDS 10% -
Sodium Azide 0.05% 0.1M
Sodium Chloride - 0.5M
Sodium Hydroxide 0.5M 2.5M
Sucrose - 30%
TCEP - 15mM
Thesit® 1% 2%
Thiourea - 2M
Tributylphosphine (TBP) 0.002M -
Tris (pH 8) 0.1M 0.5M
Triton® X-100 1% 3%
Triton® X-114 1% 3%
Tween® 20 1% 2%
Urea 4M 8M
Zwittergent® 3-12 - 1.5M
-, not tested; X, not compatible; *, two washes (optional).
Table 2: ML & CL Protein Assay are compatible with strong chaotropic extraction buffers
Buffer Composition
4M Urea, 1% SDS, 10mM EDTA, 0.8% β-Mercaptoethanol
6M Urea, 2M Thiourea, 4% CHAPS
6M Urea, 2M Thiourea, 4% NP-40
1% Sarcosyl, 0.8 β-Mercaptoethanol, 4M Guanidine Thiocyanate, 10mM EDTA
6M Urea, 2M Thiourea, 2% CHAPS, 2% ND SB 201
6M Urea, 2M Thiourea, 2% CHAPS, 2% SB 2 10
Page 6 of 13
REPONSE TO VARIOUS PROTEINS:
As with any colorimetric assay, color formation will vary between different proteins. The
graph below demonstrates narrow variation in color development between the
different proteins assayed. The ML & CL protein assays are offered with multiple choice
of protein standards.
ML PROTEIN ASSAY PROTOCOLS
(Cat. # 786-1075, 786-1076, 786-1082, 786-1083)
The ML Protein Assay is a modified Lowry version that is compatible with a wide variety
of protein samples, including samples containing detergents used in protein research
(See Table-1).
Standard ML-Protein Assay Protocol (5ml Assay Tubes)
Preparation before Use:
1. All kit components should be warmed to room temperature prior to use.
2. Prepare Reagent A: Add 20 µl of Reagent D to each 1 ml of Copper solution needed.
Each sample or standard will require 510 µl of Reagent A. Mix by gentle inversion
to prevent foaming
NOTE: If precipitate is observed in Reagent D, warm solutions to 37°C until
precipitate dissolve.
NOTE: Reagent A is stable for one week at 4°C.
NOTE: If samples do not contain detergent, you may omit this step and use Copper
solution as Reagent A.
0
1
2
3
4
5
6
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2
BSA
IgG
Ribonuclease A
Ovalbumin
Conalbumin
Protein Concentration, mg/ml
Ab
sorb
ance
Page 7 of 13
3. Prepare 4-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein.
Standards should be prepared in the same buffer as sample for best results.
Alternatively, use pre-diluted standard (Cat. # 786-1084, 786-114) and aliquot 6
dilutions from 0.1mg/ml to 1mg/ml. You would require 100 µl for each standard.
ML Protein Assay
1. Pipette 100 µl of samples and standards into each assay tubes.
2. Add 510 µl of prepared Reagent A to each tube and vortex.
3. Add 4 ml of Folin’s Reagent into each tube vortex immediately. For best result
rapidly shoot the Folin's reagent into each tube and mix immediately.
4. Incubate at room temperature for 15 minutes.
5. Read absorbance at 750 nm. The absorbance will be stable for at least 1 hour.
6. Plot absorbance against protein concentration and determine protein
concentrations of unknown samples.
Microtube ML-Protein Assay Protocol (1.5-2.0ml Assay Tubes)
Preparation before Assay
1. All kit components should be warmed to room temperature prior to use.
2. Prepare Reagent A: Add 5 µl of Reagent D to each 250 µl of Copper solution
needed. Each sample or standard will require 125 µl of Reagent A. Mix by gentle
inversion to prevent foaming
NOTE: If precipitate is observed in Reagent D, warm solutions to 37°C until
precipitate dissolve.
NOTE: Reagent A is stable for one week at 4°C.
NOTE: If samples do not contain detergent, you may omit this step and use Copper
solution as Reagent A.
3. Prepare 4-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein.
Standards should be prepared in the same buffer as sample for best results.
Alternatively, use pre-diluted standard (Cat. # 786-1084, 786-114) and aliquot 6
dilutions from 0.1mg/ml to 1mg/ml. You would require 25 µl for each standard.
ML Protein Assay
1. Pipette 25 µl of samples and standards into each assay tubes.
2. Add 125 µl of prepared Reagent A to each tube and vortex.
3. Add 1 ml of Folin’s Reagent into each tube vortex immediately. For best result
rapidly shoot the Folin's reagent into each tube and mix immediately.
4. Incubate at room temperature for 15 minutes.
Page 8 of 13
5. Read absorbance at 750 nm. The absorbance will be stable for at least 1 hour.
6. Plot absorbance against protein concentration and determine protein
concentrations of unknown samples.
Microtiter Plate ML-Protein Assay Protocol (96 well plate)
Preparation before Assay
1. All kit components should be warmed to room temperature prior to use.
2. Prepare Reagent A: Add 20 µl of Reagent D to each1 ml of Copper solution needed.
Each sample or standard will require 25 µl of Reagent A. Mix by gentle inversion to
prevent foaming
NOTE: If precipitate is observed in Reagent D, warm solutions to 37°C until
precipitate dissolve.
NOTE: Reagent A is stable for one week at 4°C.
NOTE: If samples do not contain detergent, you may omit this step and use Copper
solution as Reagent A.
3. Prepare 4-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein.
Standards should be prepared in the same buffer as sample for best results.
Alternatively, use pre-diluted standard (Cat. # 786-1084, 786-114) and aliquot 6
dilutions from 0.1mg/ml to 1mg/ml. You would require 5 µl for each standard.
ML Protein Assay
7. Pipette 5 µl of samples and standards into each assay tubes.
8. Add 25 µl of prepared Reagent A to each tube and vortex.
9. Add 200 µl of Folin’s Reagent into each tube vortex immediately. For best result
rapidly shoot the Folin's reagent into each tube and mix immediately.
10. Incubate at room temperature for 15 minutes.
11. Read absorbance at 750 nm. The absorbance will be stable for at least 1 hour.
12. Plot absorbance against protein concentration and determine protein
concentrations of unknown samples.
Page 9 of 13
CL (COMPATIBLE LOWRY) PROTEIN ASSAY PROTOCOLS
(Cat. # 786-1077, 786-1078, 786-1084, 786-1085)
The CL Protein Assay is reducing agent compatible version, suitable for protein sample
contain reducing agents (such as dithiothreitol (DTT), ß-mercaptoethanol and TCEP) and
a wide variety of commonly used laboratory agents that are known to interference with
Lowry’s protein assays (See Table-1). The CL Protein Assay uses G-Biosciences’
proprietary Universal Protein Precipitating Agent (UPPA™
).
Standard CL-Protein Assay Protocol (5ml Assay Tubes)
Preparation before Use:
1. All kit components should be warmed to room temperature prior to use.
2. Prepare Reagent A: Add 20 µl of Reagent D to each 1 ml of Copper solution needed.
Each sample or standard will require 510 µl of Reagent A. Mix by gentle inversion
to prevent foaming
NOTE: If precipitate is observed in Reagent D, warm solutions to 37°C until
precipitate dissolve.
NOTE: Reagent A is stable for one week at 4°C.
NOTE: If samples do not contain detergent, you may omit this step and use Copper
solution as Reagent A.
3. Prepare 4-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein.
Standards should be prepared in the same buffer as sample for best results.
Alternatively, use pre-diluted standard (Cat. # 786-1084, 786-114) and aliquot 6
dilutions from 0.1mg/ml to 1mg/ml. You would require 100 µl for each standard.
CL Protein Assay
1. Pipette 100 µl of samples and standards into each assay tubes.
2. Add 500 µl of UPPA™-I to each tube and vortex. Incubate the tubes at room
temperature for 1 minute.
3. Add 500 µl of UPPA™-II to each tube and vortex.
4. Centrifuge the tubes at 15,000xg for 3-5 minutes to pellet the precipitated protein.
5. Decant off the supernatant, return the tubes to the centrifuge as before, quickly
pulse to spin down residual liquid and remove with a pipette. Make sure to remove
the UPPA completely from the tube. Inverting the tube on clean and absorbent
tissue facilitate draining of the UPPA reagent.
OPTIONAL: For enhanced washing for problematic samples see the Troubleshooting
Section.
6. Add 510 µl of prepared Reagent A to each tube and vortex.
Page 10 of 13
7. Add 4 ml of Folin’s Reagent into each tube vortex immediately. For best result
rapidly shoot the Folin's reagent into each tube and mix immediately.
8. Incubate at room temperature for 15 minutes.
9. Read absorbance at 750 nm. The absorbance will be stable for at least 1 hour.
10. Plot absorbance against protein concentration and determine protein
concentrations of unknown samples.
Microtube CL-Protein Assay Protocol (1.5-2.0ml Assay Tubes)
Preparation before Use:
1. All kit components should be warmed to room temperature prior to use.
2. Prepare Reagent A: Add 5 µl of Reagent D to each 250 µl of Copper solution
needed. Each sample or standard will require 125 µl of Reagent A. Mix by gentle
inversion to prevent foaming
NOTE: If precipitate is observed in Reagent D, warm solutions to 37°C until
precipitate dissolve.
NOTE: Reagent A is stable for one week at 4°C.
NOTE: If samples do not contain detergent, you may omit this step and use Copper
solution as Reagent A.
3. Prepare 4-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein.
Standards should be prepared in the same buffer as sample for best results.
Alternatively, use pre-diluted standard (Cat. # 786-1084, 786-114) and aliquot 6
dilutions from 0.1mg/ml to 1mg/ml. You would require 25 µl for each standard.
CL Protein Assay
11. Pipette 25 µl of samples and standards into each assay tubes.
12. Add 125 µl of UPPA™-I to each tube and vortex. Incubate the tubes at room
temperature for 1 minute.
13. Add 125 µl of UPPA™-II to each tube and vortex.
14. Centrifuge the tubes at 15,000xg for 3-5 minutes to pellet the precipitated protein.
15. Decant off the supernatant, return the tubes to the centrifuge as before, quickly
pulse to spin down residual liquid and remove with a pipette. Make sure to remove
the UPPA completely from the tube. Inverting the tube on clean and absorbent
tissue facilitate draining of the UPPA reagent.
OPTIONAL: For enhanced washing for problematic samples see the Troubleshooting
Section.
16. Add 125 µl of prepared Reagent A to each tube and vortex.
17. Add 1 ml of Folin’s Reagent into each tube vortex immediately. For best result
rapidly shoot the Folin's reagent into each tube and mix immediately.
Page 11 of 13
18. Incubate at room temperature for 15 minutes.
19. Read absorbance at 750 nm. The absorbance will be stable for at least 1 hour.
20. Plot absorbance against protein concentration and determine protein
concentrations of unknown samples.
TROUBLESHOOTING SECTION
Problem Solution
Buffer used is not on the
compatibility list. Will it interfere
with the assay?
Perform a side-by-side comparison of two standard
curves:
Protein in the buffer to be tested
Protein in water
Plot protein concentration vs. absorbance. If the
buffer does not interfere, then the slopes of the
resulting standard curves will be identical. Partial
interference can be compensated for by performing
the standard curve with the buffer in question for
the actual protein assay.
Is any sample preparation
required?
No, but the protein must be solubilized.
My sample is a mixture of
proteins. Which standard should
I use for the standard curve?
A purified preparation of the target protein would
be best, but that is not always available. Any
purified protein can be selected as a reference
standard for relative protein values as long as it
gives a color yield similar to that of the protein
being assayed.
G-Biosciences offers several protein standards:
Bovine Gamma Globulin (Cat#786-007)
Bovine Serum Albumin (Cat#786-006)
Non-animal (Cat. #786-438 )
Pre-diluted standard (Cat. #786-114 )
May I use a wavelength other
than 750nm?
Yes, absorbance can be measured at 650-750 nm.
For CL Protein assay, what if
there is a failure to pellet all the
protein after 3-5 minute
centrifugation?
Centrifuge for >5 minutes and/or >15,000xg. The
protein pellet may take longer to dissolve after the
addition of Copper Solution.
Page 12 of 13
Problem Solution
For CL-Protein assay, how can I
minimize interference from
supernatant carry-over?
Readings very low and show
limited change with increasing
concentrations.
Ensure all supernatant is removed. There should be
no residual liquid. Here are a couple actions to
maximize supernatant removal:
Careful aspiration of the supernatant
Dry tubes under vacuum before
proceeding to next step
If high concentrations of interfering agents are
present, then an additional washing may be
required. A second wash can be performed as
follows: Repeat UPPA treatment steps
Reagent D developed a
precipitate when stored in
refrigerator. Can I still use it?
Yes, Reagent D will develop a precipitate during cold
room storage. Warming to 37°C for 5 minutes and
vortexing will dissolve the precipitate.
All kit components should be warmed to 25-30°C
prior to use.
REFERENCES
1. Alam, Aftab, “A Model for Formulation of Protein Assay,” Analytical Biochemistry,
203 (1992): 121-126.
2. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., “Protein Measurement
with the Folin Phenol Reagent,” Journal of Biological Chemistry, 193 (1951): 265-
275.
3. Peterson, Gary L., “Review of the Folin Phenol Protein Quantitation Method of
Lowry, Rosebrough, Farr, and Randall,” Analytical Biochemistry, 100 (1979): 201-
220.
RELATED PRODUCTS
Download our Protein Assays or Bioassay Handbook
http://info2.gbiosciences.com/complete-protein-assay-guide
http://info2.gbiosciences.com/complete-bioassay-handbook
For other related products, visit our website at www.GBiosciences.com or contact us.