ab Glucose Isomerase Activity Assay Kit (Colorimetric)

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Version 1 Last updated 1 June 2020

ab273289

Glucose Isomerase Activity Assay Kit (Colorimetric)

View Kit datasheet:

https://www.abcam.com/ab273289

(use https://www.abcam.cn/ab273289 for china, or https://www.abcam.co.jp/ab273289 for Japan)

For the measurement of Glucose Isomerase activity in bacterial lysates.

This product is for research use only and is not intended for diagnostic use.

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1. Overview

Glucose Isomerase Activity Assay Kit (Colorimetric) (ab273289) provides a quick and easy method for monitoring Glucose Isomerase (GI) activity in various samples.

For example, purified proteins and bacterial lysates (E. coli, Streptomyces murinus, etc.).

In this assay, GI converts Fructose into Glucose. This isomerization is detected through a series of enzymatic reactions generating an intermediate that reacts with the Probe producing a stable chromophore with strong absorbance at OD= 570 nm.

The assay is simple, sensitive and can detect GI activity as little as 50 µU in a variety of samples.

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ab273289 Glucose Isomerase Activity Assay Kit (Colorimetric) 2

2. Protocol Summary

Prepare samples as directed

Prepare all reagents as directed

Prepare standard curve

Add Standard, Sample, Background Control and Positive Control to appropriate wells and adjust volume to 50 µL

Add Reaction Mix to Standard, Positive Control and Samples wells.

Add Background Control Mix to Background Controls wells (50 µL)

Measure absorbance (OD = 570 nm) immediately in kinetic for 5 – 60 minutes at 37ºC.

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3. Precautions

Please read these instructions carefully prior to beginning the assay.

 All kit components have been formulated and quality control tested to function successfully as a kit.

 We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental

circumstances. However, we cannot guarantee the

performance of the product outside the conditions detailed in this protocol booklet.

 Reagents should be treated as possible mutagens and should be handled with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components.

 Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipette by mouth. Do not eat, drink or smoke in the laboratory areas.

 All biological materials should be treated as potentially

hazardous and handled as such. They should be disposed of in accordance with established safety procedures.

4. Storage and Stability

Store kit at -20°C in the dark immediately upon receipt. Kit has a storage time of 6 months from receipt.

Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual

prepared components in the Materials Supplied section.

Aliquot components in working volumes before storing at the recommended temperature.

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5. Limitations

 Assay kit intended for research use only. Not for use in diagnostic procedures.

 Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted.

6. Materials Supplied

Item Quantit

y

Storage temperatur

e (before prep)

Storage temperatur

e (after prep)

GI Assay Buffer 25 mL -20°C -20°C

GI Probe (in DMSO) 200 µL -20°C -20°C

GI Substrate 200 µL -20°C -20°C

GI Converter

(Lyophilized) 1 vial -20°C -20°C

GI Developer

(Lyophilized) 1 vial -20°C -20°C

Glucose Standard

(100 mM) 100 µL -20°C -20°C

GI Positive Control (in Ammonium Sulfate)

50 µL -20°C -20°C

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7. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to successfully perform this assay:

 Multi-well spectrophotometer capable of measuring OD at 570 nm

 Clear 96-well plate with flat bottom

 Ammonium Sulfate Solution (Saturated, 4.1 M)

 10 kDa cutoff spin filters

 Dounce homogenizer

8. Technical Hints

 This kit is sold based on number of tests. A “test” simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions.

 Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures.

 Avoid foaming or bubbles when mixing or reconstituting components.

 Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.

 Ensure plates are properly sealed or covered during incubation steps.

 Ensure all reagents and solutions are at the appropriate temperature before starting the assay.

 Samples generating values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer.

 Make sure all necessary equipment is switched on and set at the appropriate temperature.

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9. Reagent Preparation

Briefly centrifuge small vials at low speed prior to opening.

9.1 GI Assay Buffer:

Ready to use as supplied. Store at -20 °C. Bring to room temperature before use.

9.2 GI Substrate:

Ready to use as supplied. Store at -20 °C. Bring to room temperature before use.

9.3 GI Converter (Lyophilized):

Reconstitute with 220 µL GI assay buffer. Pipette up and down to dissolve completely. Store at -20°C. Use within two months.

9.4 GI Developer (Lyophilized):

Reconstitute with 220 µL GI assay buffer. Pipette up and down to dissolve completely. Store at -20°C. Use within two months.

9.5 GI Probe (in DMSO):

Ready to use as supplied. Warm to room temperature prior to use. Store at –20°C, protect from light and moisture. Use within two months.

9.6 Glucose Standard (100 mM):

Store at -20°C. Thaw at room temperature before use.

9.7 GI Positive Control (in Ammonium Sulfate):

Note: GI is extremely unstable when not in (NH4)2SO4 Solution.

Mix GI Positive Control well and take a 10 μL GI aliquot;

centrifuge aliquot at 10,000 x g for 5 min at room temperature.

Carefully remove the supernatant and dissolve pellet with 100 µL Assay Buffer. Mix thoroughly. Use this solution within 4 hours. Store rest of GI Positive Control in (NH4)2SO4 solution at 4°C. Use within two months.

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10.Sample Preparation

Bacterial lysate:

10.1 Grow bacteria at desired conditions, collect culture and spin down at 10,000 x g, 20 minutes at 4°C.

10.2 Remove the supernatant and homogenize pellets (100 mg) with 1 mL ice-cold GI Assay Buffer using homogenizer for 2 minutes on ice, and place on ice for 10 minutes.

10.3 Centrifuge at 10,000 x g for 10 minutes at 4°C.

10.4 Collect the supernatant.

10.5 Use ammonium sulfate precipitation to remove interferences:

Aliquot tissue samples (100 µL) to clean centrifuge tubes, add saturated 4.32 M ammonium sulfate reaching 65% saturation (1 volume of sample + 2 volumes of 4.32 M ammonium sulfate). Place samples on ice for 30 minutes. Spin down samples at 10,000 x g at 4°C for 10 minutes, discard the supernatant, and resuspend the pellet back to the original volume with GI Assay Buffer.

Note: If you would like to determine specific GI activity in the samples use a BCA Reducing Agent Compatible Protein Assay Kit or similar, to determine protein concentration in samples.

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11.Standard Curve

11.1 Prepare a 1 nmol/µl (1 mM) solution of Glucose Standard by diluting 10 µL of 100 mM Glucose Standard with 990 µL of GI Assay Buffer.

11.2 Mix well. Add 0, 2, 4, 6, 8 and 10 μL of the 1 mM Glucose Standard into a series of wells in a clear 96-well plate to obtain 0, 2, 4, 6, 8 and 10 nmol/well.

11.3 Adjust the volume of each well to 50 μL with GI Assay Buffer.

Standard # Glucose Standard (µL)

GI Assay Buffer (µL)

Glucose Standard (nmol/well)

1 10 40 10

2 8 42 8

3 6 44 6

4 4 46 4

5 2 48 2

6 0 50 0

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12.Assay Procedure

 Thaw all reagents thoroughly and mix gently.

 For unknown samples, we suggest testing several concentrations to ensure the readings are within the Standard Curve range.

 To control for sample background, prepare parallel sample wells as sample background controls.

12.1 For Sample (S), prepare two wells for each sample labeled

“Sample Background Control” (BC), and “Sample” (S).

Add 2 -50 μL sample into each of these wells.

12.2 For Positive Control, add 2 - 20 µL of the provided GI Positive Control.

12.3 Adjust the volume of Sample, Sample Background Control and Positive Control to 50 μL/well with GI Assay Buffer. Mix well.

12.4 Reaction Mix: Prepare enough reagents for the number of assays to be performed. For each well, prepare 50 μL of Reaction Mix. Mix well.

Component Reaction Mix Background Control

Mix

GI Assay Buffer 42 μL 44 μL

GI Converter 2 μL 2 μL

GI Developer 2 μL 2 μL

GI Probe 2 μL 2 μL

GI Substrate 2 μL ---

12.5 Add 50 µL of the Reaction Mix to each well containing the Standard, Positive Control and Samples [S] and 50 µL of Background Control Mix to each well containing the Sample Background Controls [BC]. Mix well.

12.6 Measure absorbance immediately at OD = 570 nm in kinetic mode for 5-60 minutes at 37ºC.

Note: Incubation time depends on the Glucose Isomerase activity in the samples. We recommend measuring the OD in a kinetic mode

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13.Calculations

13.1 Subtract the 0 standard reading from all standard readings.

13.2 Plot the Glucose standard curve.

13.3 Correct sample reading by subtracting the value derived from the sample background control from all sample readings.

13.4 Calculate the GI activity of the test sample: ∆OD = A₂ – A₁. 13.5 Apply the ∆OD to the Glucose standard curve to get B nmol of

Glucose generated by Glucose Isomerase during the reaction time (∆t = t2 - t1)

13.6 GI activity may be calculated using the following equation:

𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝐼𝑠𝑜𝑚𝑒𝑟𝑎𝑠𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝐵

(Δ𝑡 𝑥 𝑉)𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 = 𝑛𝑚𝑜𝑙/𝑚𝑖𝑛/𝑚𝐿 = 𝑚𝑈/𝑚𝐿

B = Glucose amount from standard curve (nmol).

Δt = Reaction time t2 – t1 (min).

V = Sample volume added into the reaction well (mL).

Unit Definition: One unit of Glucose Isomerase is the amount of enzyme that isomerizes 1.0 µmol of glucose per minute at pH 8.0 at 37ºC.

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14.Typical Data

Typical standard curve – data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.

Figure 1. Glucose standard curve.

Figure 2. Kinetic measurement of Glucose Isomerase activity from Streptomyces lysates (µg protein):Sample A (26 µg), Sample B (52 µg).

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ab273289 Glucose Isomerase Activity Assay Kit (Colorimetric) 12 Figure 3. Relative GI activity was calculated in lysates prepared from E. coli.

and Streptomyces. Assays were performed following kit protocol.

15.FAQ / Troubleshooting

General troubleshooting points are found at www.abcam.com/assaykitguidelines.

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16.Notes

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Technical Support

For all technical or commercial enquiries please go to:

www.abcam.com/contactus www.abcam.cn/contactus (China) www.abcam.co.jp/contactus (Japan)