ml/cl protein assays - g-biosciences · g-biosciences’ ml & cl protein assays are two...

G-Biosciences 1-800-628-7730 1-314-991-6034 [email protected]

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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)

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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

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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.

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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

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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

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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

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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


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










ML Protein Assay






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Microtiter Plate ML-Protein Assay Protocol (96 well plate)

Preparation before Assay


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










ML Protein Assay



9. Add 200 µl of Folin’s Reagent into each tube vortex immediately. For best result






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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)



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










CL Protein Assay


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.


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Microtube CL-Protein Assay Protocol (1.5-2.0ml Assay Tubes)



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










CL Protein Assay


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.




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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.

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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.




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ml/cl protein assays - g-biosciences · g-biosciences’ ml & cl protein assays are two...

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