6-Phosphofructokinase Activity Assay Kit (Colorimetric)

Transcription

1 ab Phosphofructokinase Activity Assay Kit (Colorimetric) Instructions for Use For the sensitive and accurate measurement of phosphofructokinase activity in animal tissue and cell culture samples (adherent and suspension) This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis Troubleshooting 12 2

4 1. Overview Phosphofructokinase (PFK) is a key glycolytic enzyme and plays a major regulatory role during glycolysis. This enzyme is present in bacteria, plants and animals. There are 2 types of PFKs - PFK1 and PFK2. In the presence of ATP, PFK1 & PFK2 catalyzes the conversion of fructose-6-phosphate to fructose-1,6-diphosphate and fructose-2,6-diphosphate respectively and ADP. PFK has 3 major isoforms in mammals: PFK-M (muscle), PFK-L (liver) and PFK-P (platelet). In humans, PFK deficiency causes glycogen storage disease, also called Tarui s disease, which is characterized by exercise-induced muscle weakness and cramps. On the other hand, increased PFK activity contributes to cancer cell proliferation and tumorigenicity. Early detection of abnormal phosphofructokinase activity is crucial for diagnosis, prediction and therapeutic strategy. In Abcam's 6-Phosphofructokinase Activity Assay kit (ab155898), PFK converts fructose-6-phosphate and ATP to fructosediphosphate and ADP. The ADP in the presence of substrate and enzyme mix is converted to AMP and NADH, which reduces a colorless probe to a colored product with strong absorbance at 450 nm. PFK activity assay is simple, robust, and rapid and can detect phosphofructokinase activity less than 1 mu. 3

5 2. Protocol Summary Reagent Preparation Standard Curve Preparation Sample Preparation Positive Control Reaction Mix Measurement and Calculation 4

6 3. Components and Storage A. Kit Components Item Quantity Assay Buffer 27 ml PTK Substrate (Lyophilized) 1 vial ATP (Lyophilized) 1 vial Enzyme Mix (Lyophilized) 1 vial Developer (Lyophilized) 1 vial NADH Standard (Lyophilized) 1 vial Positive Control (Lyophilized) 1 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

7 B. Additional Materials Required 96-well clear plate with flat bottoms Multi-well spectrophotometer (ELISA reader) 6

8 4. Assay Protocol A. Reagent Preparation 1. PFK Substrate: Reconstitute with 220 μl Assay Buffer. Store at -20 C. Use within two months. Keep on ice while in use. 2. ATP: Reconstitute with 220 μl dh 2 O. Store at -20 C. Use within two months. Keep on ice while in use. 3. 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. Use within two months. Keep on ice while in use. 4. Developer: Reconstitute with 220 μl dh 2 O. Pipette up and down to dissolve completely. Store at -20 C. Use within two months. 5. NADH Standard: Reconstitute with 40 μl Assay Buffer to generate 10 mm NADH Standard stock solution. Store at 20 C. Use within two months. Keep on ice while in use. 7

9 6. Positive Control: Reconstitute with 100 μl Assay Buffer and mix thoroughly. Aliquot and store at 20 C. B. Phosphofructokinase Activity Assay Protocol 1. NADH Standard Curve: Dilute NADH Standard 10 fold by adding 10 μl NADH Standard stock solution to 90 μl Assay Buffer. Add 0, 2, 4, 6, 8 and 10 μl of 1 mm diluted NADH Standard into a series of wells in 96 well plate to generate 0, 2, 4, 6, 8 and 10 nmol/well of NADH Standard. Adjust final volume to 50 μl/well with Assay Buffer. 2. Sample Preparation: Rapidly homogenize tissue (20 mg) or cells (2 x 10 6 ) with 200 μl ice cold Assay Buffer for 10 minutes on ice. Centrifuge at rpm for 5 min. Collect the supernatant. Add 1-50 μl sample (100 μg) per well, adjust final volume to 50 μl with Assay Buffer. Prepare a parallel sample well as the background control to avoid interference from ADP and NADH in the sample. Note: a) For unknown samples, we suggest testing several doses to ensure the readings are within the standard curve range. 8

10 b) Small molecules in some samples such as liver may interfere with PFK Activity Assay. ADP, NADH and other interfering small molecules can be removed by 10 KD spin column. 3. Positive Control: Add μl of diluted PFK Positive Control into well(s). Adjust final volume to 50 μl with Assay Buffer. 4. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare 50 μl Reaction Mix containing: Reaction Mix Background Control Mix Assay Buffer 42 µl 44 µl Enzyme Mix 2 µl 2 µl Developer 2 µl 2 µl ATP 2 µl 2 µl PTK Substrate 2 µl --- Add 50 μl of the Reaction Mix to each well containing the Standard, Positive Control and test samples and 50 μl of Background Control mix to each well containing the Background Control sample. Mix well. 9

11 5. Measurement: Incubate for min at 37 0 C and measure OD450nm. Note: Incubation time depends on the Phosphofructokinase activity in the samples. We recommend measuring the OD in a kinetic mode, and choose two time points (T1 & T2) in the linear range to calculate the PFK activity of the samples. The NADH standard curve can read in Endpoint mode (i.e., at the end of incubation time). 5. Data Analysis Calculation: Subtract the 0 standard reading from all standard readings. Plot the NADH standard curve. Correct sample background by subtracting the value derived from the background control from all sample readings. Calculate the hexokinase activity of the test sample: ΔOD = A2 A1. Apply the ΔOD to the NADH standard curve to get B nmol of NADH generated by Phosphofructokinase during the reaction time (ΔT = T2 - T1). Sample Phosphofructokinase Activity B = (ΔT x V) x Dilution Factor = nmol/min/ml/ = mu/ml Where: B is the NADH amount from standard curve (nmol). ΔT is the reaction time (min). V is the sample volume added into the reaction well (ml). 10

12 Unit Definition: One unit of phosphofructokinase is the amount of enzyme that will generate 1.0 μmol of NADH per min at at ph 7.4 at 37 0 C. Figure 1: NADH standard curve (a). Phosphofructokinase activity in bovine liver and HeLa cell lysate (b). Assays were performed following kit protocol. 11

13 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 12

14 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) 13

15 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 (technical@abcam.com) or phone (select contact us on for the phone number for your region). 14

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