ab176722 Glucose 6 Phosphate Dehydrogenase Assay Kit (Fluorometric) Instructions for Use For monitoring Glucose 6 Phosphate Dehydrogenase in a variety of biological samples. This product is for research use only and is not intended for diagnostic use. Version: 1 Last Updated: 31 October 2013
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Table of Contents Table of Contents 2 1. Overview 3 2. Protocol Summary 5 3. Kit Components 6 4. Storage and Stability 7 5. Materials Required, Not Supplied 7 6. Assay Protocol 8 7. Data Analysis 10 8. Troubleshooting 12 2
1. Overview Glucose 6 Phosphate Dehydrogenase (G6PD) catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone, the first and rate-limiting step in the pentose phosphate pathway. It is critical metabolic pathway that supplies reducing energy to cells (such as erythrocytes) by maintaining the level of co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH), and for the production of pentose sugars. The production of NADPH is of great importance for tissues actively engaged in biosynthesis of fatty acids and/or isoprenoids, such as the liver, mammary glands, adipose tissue, and the adrenal glands. The NADPH also maintains the level of glutathione in these cells that helps protect the red blood cells against oxidative damage. Deficiencies in G6PD predispose individuals to non-immune hemolytic anemia. Abcam's Glucose 6 Phosphate Dehydrogenase Assay Kit (Fluorometric) (ab176722) provides a simple, sensitive and rapid fluorescence-based method for detecting G6PD in biological samples such as serum, plasma, urine, as well as in cell culture samples. In the enzyme coupled assay, G6PD activity is proportionally related to the concentration of NADPH that is specifically monitored by a fluorogenic NADPH sensor to yield a highly red fluorescence product. The fluorescence signal can be read with a fluorescence microplate reader at Ex/Em = 540 nm/590 3
nm. With the G6PD assay kit, we were able to detect as little as 0.3 mu/ml G6PD in a 100 µl reaction volume. It is robust, and can be readily adapted for a wide variety of applications that require the measurement of G6PD. 2. Protocol Summary Prepare G6PD assay mixture Add G6PD standards or test samples Incubate at RT for 30 min 2 hours Monitor fluorescence intensity at Ex/Em = 540/590 nm 4
3. Kit Components Item Quantity Storage upon arrival Storage after use/ reconstitution Enzyme Probe (lyophilized) 2 bottles -20 C -20 C Assay Buffer 10 ml -20 C -20 C NADP 1 vial -20 C -20 C G6PD Standard 10U 1 vial -20 C -20 C 4. Storage and Stability Upon arrival, store the kit at-20 C and protected 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
5. Materials Required, Not Supplied 96 or 384-well black plate with clear flat bottoms Multi-well spectrophotometer (ELISA reader) Distilled water or MilliQ PBS 6
6. Assay Protocol 1. Reagent Preparation a) Prepare NADP stock solution (100X): Add 100 μl of H 2 O into the vial of NADP to make 100X NADP stock solution. Mix well. b) Prepare G6PD stock solution: Add 100 μl of H2O or 1X PBS buffer into the vial of G6PD Standard (Component D) to make 100 U/mL G6PD standard solution. NOTE: The unused G6PD standard stock solution should be divided into single use aliquots and stored at -20 C. 2. Prepare serial dilutions of G6PD standard (0 to 300 mu/ml): a) Add 10 μl of G6PD stock solution into 990 μl 1X PBS buffer to generate 1000 mu/ml G6PD standard solution. NOTE: Diluted G6PD standard solution is unstable, and should be used within 4 hours. b) Take 200 μl of 1000 mu/ml G6PD standard solution to perform 1:3 serial dilutions to get 300, 100, 30, 10, 3, 1, 0.3, and 0 mu/ml serial dilutions of G6PD standard. c) Add serial dilutions of G6PD standard and G6PD containing test samples into a solid black 96-well microplate as described in the tables below. 7
BL BL TS TS.. G6PD 1 G6PD 1.... G6PD 2 G6PD 2 G6PD 3 G6PD 3 G6PD 4 G6PD 4 G6PD 5 G6PD 5 G6PD 6 G6PD 6 G6PD 7 G6PD 7 NOTE: G6PD = Glucose 6 Phosphate Dehydrogenase Standards, BL = Blank Control, TS = Test Samples. Reagent composition for each well Pyrophosphate Standards Blank Control Test Sample Serial Dilutions*: 50 μl Assay Buffer: 50 μl 50 μl Note: * Add the serially diluted G6PD standards from 0.3 mu/ml to 300 mu/ml into wells from G6PD1 to G6PD7 in duplicate. 3. Prepare G6PD assay mixture: a) Add 5 ml of Assay Buffer into one bottle of Enzyme Probe. b) Add 50 µl NADP stock solution (100X) into the bottle of Enzyme Probe and mix well. NOTE: This G6PD assay mixture is enough for one 96-well plate. The unused assay mixture should be divided into single use aliquots and stored at -20 C. 8
4. Run G6PD assay a) Add 50 μl of G6PD assay mixture to each well of G6PD standard, blank control, and test samples to make the total assay volume of 100 µl/well. NOTE: For a 384-well plate, add 25 μl of sample and 25 μl assay mixture into each well. 7. Data Analysis The fluorescence in blank wells (with the dilution buffer only) is used as a control, and is subtracted from the values for those wells with the G6PD reactions. A G6PD standard curve is shown in Figure 1. Note: The fluorescence background increases with time, thus it is important to subtract the fluorescence intensity value of the blank wells for each data point. 9
Figure 1. G6PD dose response was measured with Abcam s Glucose 6 Phosphate Dehydrogenase Assay Kit (Fluorometric) (ab176722) in a 96-well black plate using a microplate reader. As low as 0.3 mu/ml glucose 6 phosphate dehydrogenase in 100 µl volume can be detected with 1 hour incubation. 10
8. 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 11
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) 12
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 13
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