ab136955 Glucose Uptake Colorimetric Assay Kit Instructions for Use For the sensitive and accurate measurement of Glucose uptake in various samples This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 11 6. Troubleshooting 13 2
1. Overview Glucose uptake is an important biological process for studying cell signaling and glucose metabolism. Among many different methods available for measuring glucose uptake, 2-deoxyglucose (2-DG) has been widely used because of its structural similarity to glucose. As with glucose, 2-DG can be taken up by glucose transporters and metabolized to 2-DG-6-phosphate (2-DG6P). 2-DG6P, however, cannot be further metabolized, and thus accumulates in the cells. The accumulated 2-DG6P is directly proportional to 2-DG (or glucose) uptake by cells. In Abcam s glucose uptake colorimetric assay kit, the 2-DG6P is oxidized to generate NADPH, which can be determined by an enzymatic recycling amplification reaction. This easy to use nonradioactive kit is highly sensitive and can detect glucose uptake as low as 10 pmol/well. It can be used for the measurement of glucose uptake in response to insulin, growth factors, cytokines, mitogens, and nutrients, etc., the analysis of glucose metabolism and cell signaling in various cell types and the screening of anti-diabetic drugs. 3
Figure 1: Assay Procedure. Step A: 2-DG oxidation to generate NADPH; Step B: NADPH recycling amplification Reaction. 2. Protocol Summary Reagent Preparation Standard Curve Preparation Sample Preparation Reaction Measurement and Calculation 4
3. Components and Storage A. Kit Components Item Quantity Extraction Buffer 17 ml Neutralization Buffer 2.5 ml 2-Deoxyglucose (2-DG, 10 mm) 1 ml Assay Buffer 10 ml Enzyme Mix (Lyophilized) 1 vial Recycling Mix(Lyophilized) 1 vial 2-DG6P Standard (Lyophilized) 1 vial Glutathione Reductase (Lyophilized) 2 vial Substrate-DTNB (Lyophilized) 2 vial * Store the kit at-20 C and protect from light. Please read the entire protocol before performing the assay. Avoid repeated freeze/thaw cycles. Warm all Buffers to room temperature before use. Briefly centrifuge all small vials prior to opening. 5
B. Additional Materials Required 96-well clear plate with flat bottoms Plate sealing tape Multi-well spectrophotometer (ELISA reader) Multi-channel pipette KRPH buffer: 20 mm Hepes, 5 mm KH 2 PO 4, 1 mm MgSO 4, 1 mm CaCl 2, 136 mm NaCl, 4.7 mm KCl, ph 7.4. 6
4. Assay Protocol A. Reagent Preparation 1. Enzyme Mix: Reconstitute with 220 μl Assay Buffer. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Avoid repeated freeze/thaw cycles. 2. Recycling Mix: Reconstitute with 220 μl Assay Buffer. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Avoid repeated freeze/thaw cycles. 3. 2-DG6P Standard: Reconstitute with 100 μl dh 2 O to generate a 10 mm (10 nmol/μl) 2-DG6P Standard solution. Keep on ice while in use. Store at 20 C. Use within two months. 4. Glutathione Reductase: Reconstitute with 1.1 ml Assay Buffer. Mix well, aliquot and store at 20 C. Avoid repeated freeze/thaw cycles. 5. Substrate-DTNB: Reconstitute with 1 ml Assay Buffer. Dissolve completely by pipetting up and down. Store at 20 C. Reconstituted substrate is stable for 2 months. 7
B. Glucose Uptake Assay Protocol 1. 2-DG6P Standard Curve: Dilute 2-DG6P Standard to 0.1 mm (100 pmol/μl) by adding 10 μl of 10 mm 2-DG6P to 990 μl Assay Buffer and mix well. Dilute further to 0.01 mm (10 pmol/μl) by adding 50 μl of 0.01 mm 2-DG6P to 450 μl Assay Buffer. Add 0, 2, 4, 6, 8 and 10 μl into a series of well on a 96 well plate in duplicate to generate 0, 20, 40, 60, 80 and 100 pmol/well of 2-DG6P standard. Adjust volume to 50 μl/well with Assay Buffer. Note: make fresh dilution with the 10mM 2-DG6P standard stock solution each time. 2. Sample Preparation: Treat cells with desired method. For 3T3-L1 adipocytes, for example, seed cells at a density of ~1500 cells per well in a 96 well plate and differentiate to mature adipocytes, maintain for another 4 days prior to use. To assay glucose uptake, wash adipocytes twice with PBS and starve in 100 μl serum free adipocyte medium overnight (to increase glucose uptake). Wash cells 3X with PBS. Starve the cells for glucose by preincubating with 100 μl Krebs-Ringer-Phosphate-Hepes (KRPH) buffer containing 2 % BSA for 40 min. Stimulate with or without insulin (1 μm) for 20 min to activate glucose transporter. Add 10 μl of 10 mm 2-DG, mix and incubate for 20 min. 8
Note: As a control, prepare a parallel sample well not treated with insulin and 2-DG. Wash cells 3X with PBS to remove exogenous 2- DG. To degrade endogenous NAD(P) and to denature enzymes, lyse cells with 80 μl of Extraction Buffer, freeze/thaw once and heat at 85 C for 40 min. Cool the cell lysate on ice for 5 min and neutralize by adding 10 μl of Neutralization Buffer. Briefly spin & dilute 1:10 times by adding 45 μl of Assay Buffer to 5 μl sample. Add 1-50 μl sample per well, adjust final volume to 50 μl with Assay Buffer. Notes: For other cell types, optimal incubation times and treatment protocols may vary from these conditions. We suggest testing several doses of cell lysate to ensure the readings are within the standard curve range. We suggest starving cells overnight in serum free medium to increase 2-DG uptake. 3. 2-DG6P Standard: a. NADPH Generation: Mix enough reagents for the number of assays (Control, samples and standards) to be performed. For each well, prepare 10 μl Reaction Mix A: Reaction Mix A Assay Buffer 8 µl Enzyme Mix 2 µl Mix. Add 10 μl Reaction Mix A into each well. Mix. Incubate at 37 C for 1 hour. 9
b. NADP Degradation: To degrade unused NADP (Figure 1, step A), add 90 μl of extraction buffer to each well, seal with aluminum sealing tape and heat at 85 C for 40 min. Cool on ice for 5 min and add 12 μl of neutralization buffer. c. Recycling Amplification Reaction: For each well (control, standards and samples), prepare 38 μl recycling reaction mix B: Reaction Mix B Glutathione Reductase 20 μl Substrate (DTNB) 16 μl Recycling Mix 2μl Mix. Add 38 μl Reaction Mix B into each well. Mix. Incubate at 37 C for 40 minutes. 4. Measurement: Measure absorbance at 412 nm in microplate reader. 10
5. Data Analysis Calculation: Subtract the 0 pmol standard from all standard readings. Plot the 2-DG6P Standard Curve. Correct sample background by subtracting the value derived from the untreated cells (Control i.e. not treated with insulin and 2-DG) from all sample readings (Note: The background reading can be significant and must be subtracted from all sample readings). 2-DG concentration of the test samples, which is proportional to accumulated 2-DG6P can then, be calculated. 2-DG uptake = Sa/Sv (pmol/μl or nmol/ml or μm) Where: Sa is the amount of 2-DG6P (in pmol) in sample well calculated from Standard Curve. Sv is sample volume (in μl) added into the sample well. 11
Figure. 2. (a) 2-DG6P Standard curve (b), (c) and (d) 2-DG uptake in 3T3-L1 cells, Human adipocytes and HeLa cells respectively. I=Insulin; P=Phloretin. 12
6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 13
Problem Reason Solution Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 14
Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 15
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