Enzymatic Method for Determining Glutamate (Glutamate Assay)

This assay protocol is suitable for the colorimetric detection of glutamate in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Glutamate Assay Kit (MAK004). Glutamate concentration is determined by an enzymatic assay, which results in a colorimetric (450 nm) product, proportional to the glutamate present. The linear range of detection for this assay is between 2–10 nmole.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

Reagents

Glutamate Assay Buffer                      25 mL
    Catalog Number MAK004A

Glutamate Enzyme Mix                      1 vl
    Catalog Number MAK004B

Glutamate Developer                          1 vl
    Catalog Number MAK004C

Glutamate Standard, 0.1 M                0.1 ml   
    Catalog Number MAK004D

Reagents and Equipment Required but Not Provided

96 well flat-bottom plate – It is recommended to use clear plates for colorimetric assays (Catalog Number M4436 or equivalent).

Spectrophotometric multiwell plate reader

10 kDa Molecular Weight Cut-Off (MWCO) Spin Filter (Catalog Number Z706345 or equivalent)

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Glutamate Assay Buffer – Allow buffer to come to room temperature before use.

Glutamate Enzyme Mix – Reconstitute in 220 µL of Glutamate Assay Buffer. Mix well by pipetting. Aliquot enough of reconstituted Enzyme Mix for use in assay (2 µL per assay well), and then immediately aliquot and store the remainder at –20 °C. Keep the enzyme mix on ice until use in assay. The enzyme mix is stable for at least
2 months after reconstitution when stored at –20 °C in a frost-free freezer.

Glutamate Developer – Reconstitute in 820 µL water. Mix well by pipetting (do not vortex). Aliquot and store at –20 °C. Use within 2 months of reconstitution.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate.

Procedure

Glutamate Standards for Colorimetric Detection
Dilute 10 mL of the 0.1 M Glutamate Standard with 990 µL of the Glutamate Assay Buffer to prepare a 1 mM standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the 1 mM standard solution into a 96 well plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Glutamate Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation
Tissue or cells (1 x 106) can be homogenized in 100 µL of the Glutamate Assay Buffer. Centrifuge the samples at 13,000 x g for 10 minutes to remove insoluble material. Serum samples (10–50 µL) can be directly added to wells.

Samples may be deproteinized with a 10 kDa MWCO spin filter prior to addition to the reaction. This step may be necessary if enzymes in the samples interfere with the assay. If protein, fat, or solids/particulates are present, samples should be filtered through a 10 kDa MWCO spin filter.

Bring samples to a final volume of 50 µL with Glutamate Assay Buffer.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

Include a blank sample for each set of samples by omitting the Glutamate Enzyme Mix in the Reaction Mix.

Assay Reaction
1.     Set up the Reaction Mixes according to the scheme in Table 1. 100 µL of Reaction Mix is required for each reaction (well).

Table 1.
Reaction Mixes

Reagent Blank Sample Samples and Standards
Glutamate Assay Buffer 92 µL 90 µL
Glutamate Developer 8 µL 8 µL
Glutamate Enzyme Mix - 2 µL

2.     Add 100 µL of the appropriate Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting and incubate the reaction for 30 minutes at 37 °C. Protect the plate from light during the incubation.

3.     Measure the absorbance at 450 nm (A450).

Results

Calculations
The background for the assays is the value obtained for the 0 (blank) glutamate standard. Correct for the background by subtracting the blank value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate glutamate standards to plot a standard curve.
Note: A new standard curve must be set up each time the assay is run.

Subtract the blank sample reading from the sample readings to obtain the corrected measurement for each sample. Using the corrected measurement, the amount of glutamate present in the sample may be determined from the standard curve.

Concentration of Glutamate

        Sa/Sv = C

Sa = Amount of glutamate in unknown sample (nmole) from standard curve
Sv = Sample volume (mL) added into the wells
C = Concentration of glutamate in sample

Glutamate molecular weight: 147.3 g/mole

Sample Calculation
Amount of glutamate (Sa) = 5.84 nmole
(from standard curve)
Sample volume (Sv) = 50 µL

Concentration of glutamate in sample

5.84 nmole/50 µL = 0.1168 nmole/µL

0.1168 nmole/µL x 147.3 ng/nmole= 17.2 ng/µL

MAK004-Standard-Curve

Materials

     
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