Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit

Transcription

1 PROTOCOL Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit DESCRIPTION Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit Sufficient materials are provided for 96 measurements (one 96-well microplate) 1850 Millrace Drive, Suite 3A Eugene, Oregon MS740 Rev.1 Kit Contents: Item Quantity Detergent (10X) 1 ml Blocking reagent (10X) 10 ml Buffer (20X) 25 ml Detector (20X) 1 ml AP Label (20X) 1 ml Development buffer (1X) 20 ml Development reagent (50X) 0.4 ml 96-well Microplate (12 strips) 1 The 96-well microplate in this kit has a monoclonal antibody pre-bound to the wells. This plate can be broken into 12 separate 8-well strips; therefore the plate can be used for up to 12 separate experiments. INTRODUCTION The Mitochondrial Trifunctional Protein (TFP, Swiss-Prot entries P40939 and P55084) is part of the mitochondrial Fatty Acid β-oxidation pathway. It is located in the inner mitochondrial membrane and it catalyzes three out of the four steps in the β-oxidation cycle with a specific affinity for long chain substrates. TMP is hetero-octamer composed of four α and four β-subunits. The α-subunit is a 79 kda enzyme which has two enzymatic functions, the hydration of enoyl-coa (E.C ) and the dehydrogenation of 3-hydroxyacyl CoA (LCHAD E.C ). The β-subunit, a 47 kda enzyme, performs the third function - the thiolytic cleavage of 3-ketoacyl-CoA substrates (E.C ). Inherited Deficiency of TFP has been described as either an isolated deficiency of the dehydrogenase activity (LCHAD) with normal or partially reduced activity of the hydratase and the thiolase activities, or a complete deficiency of all three components. Heterozygous individuals for TMP defects are more susceptible to nonalcoholic fatty liver disease, the most common form of liver disease in the United States. The Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit (Catalog # MS740) is used to determine the amount of TFP protein in a sample by a sandwich ELISA assay. The enzyme is captured in the wells between two monoclonal antibodies binding to different epitopes. This sandwich is labeled with a goat anti-mouse antibody conjugated to alkaline phosphatase. The phosphatase generates a change in the color of the reaction in a time dependant manner. The color change is proportional to the amount of protein captured in the well and can be measured in the spectrophotometer at 405 nm.

2 The protocol has 4 steps: A) Sample preparation B) Plate loading C) Measurement D) Data Analysis This assay is designed for use with purified mitochondria, homogenized skeletal muscle and skin fibroblasts cell extracts. For other tissues or cell types, sample optimization may be necessary so that measurements are carried out within the linear range. For sample loading, use the maximum amount shown in the linear range below. A control or normal sample should always be included in the assay as reference. It is suggested to perform a standard curve with this control sample. Also, include a null or buffer control to act as a background reference measurement. Typical linear ranges: Cultured primary cell extracts HepG2 cell extracts Tissue extracts Tissue mitochondria (Heart and Liver) 2-20 µg/well 1-10 µg/well µg/well µg/well Typical intra-assay variations (same day, same sample) are < 10% ADDITIONAL MATERIALS REQUIRED Spectrophotometer measuring absorbance of 405 nm (The SPECTRAmax from Molecular Devices is recommended.) Method for determining protein concentration Deionized water (dh 2 O) 1X Phosphate Buffered Saline (1X PBS) Protease Inhibitors Multichannel pipette Page 2 of 9

3 MICROPLATE ASSAY PROTOCOL NOTE: This protocol contains detailed steps for measuring mitochondrial TFP protein content. Be completely familiar with the protocol before beginning the assay. Do not deviate from the specified protocol steps or optimal results may not be obtained. A. Sample Preparation 1. Cultured primary fibroblasts a. Freeze 1x10 7 cells in a dry pellet at -80 C. b. Thaw the pellet and resuspend in 200 µl of cold 1X PBS with protease inhibitors (not provided in the kit). If using more or less cells, resuspend in a proportional volume. c. Add 1/10 th volume of Detergent to the cell suspension and mix thoroughly (e.g. if the total sample is 200 µl add 20 µl of Detergent). d. Place on ice for 30 minutes. e. Centrifuge in tabletop microphage at maximum speed (~25,000 g) for 20 minutes at 4 C. f. Collect supernatant and save as extract. Discard the pellet. g. Measure protein concentration and keep sample on ice until ready to proceed. 2. Mitochondria a. Make sure that the mitochondria preparation is at a concentration of 5.5 mg/ml of protein. Adjust concentration with the same buffer used for the mitochondria isolation. b. Add 1/10 th volume of 10X Detergent to the sample and mix thoroughly. c. Place on ice for 30 minutes. d. Centrifuge in tabletop microfuge at maximum speed (~25,000 g) for 20 minutes at 4 C. e. Collect supernatant and save as extract. Discard the pellet. f. Measure protein concentration and keep sample on ice until ready to proceed. 3. Tissue Homogenate a. Dissect mg of tissue and keep cold inside a microfuge tube. It is recommended to use a homogenizer with a dispersing tool of 5 mm diameter or less such as the IKA Ultra Turrax T8. b. Add 10 times in volume of cold 1XPBS with protease inhibitors (not provided in the kit) (e.g. for 50 mg add 500 µl of volume). c. Add 1/10 th volume of 10X Detergent to the suspension. d. Homogenize on ice with 5 second bursts until no clumps are observed macroscopically. e. Keep on ice for 30 minutes. f. Centrifuge in tabletop microfuge at maximum speed (~25,000 g) for 20 minutes at 4 C. g. Collect supernatant and save as extract. Discard the pellet. h. Measure protein concentration and keep sample on ice until ready to proceed. Page 3 of 9

4 B. Plate Loading NOTE: When pipetting onto the plate avoid dripping from tips and pressing tips onto wells. When emptying wells, turn the plate upside down and in a rapid downward vertical motion force the liquid out of the wells. 1. Prepare Wash as follows: 25 ml 20X Buffer ml deionized H 2 O Keep at room temperature during the course of the assay. Store at 4 C for future use. 2. Prepare Dilution Buffer as follows: 90 ml of Wash + 10 ml 10X Blocking Reagent This is enough for a 96 well assay. If using fewer wells, adjust accordingly. Keep at room temperature during the course of the assay. Always make fresh. 3. Dilute sample extracts in Dilution Buffer at the following recommended concentrations: Cultured primary cell extracts HepG2 cell extracts Tissue extracts Tissue mitochondria (heart and liver) 100 µg/ml (~20 µg/200 µl) 50 µg/ml (~10 µg/200 µl) 25 µg/ml (~5 µg/200 µl) 1.25 µg/ml (~0.25 µg/200 µl) 4. Add 200 µl of each diluted sample into individual wells on the plate. Include a normal sample as a positive control. Include a buffer control (200 µl Dilution Buffer) as a background reference. 5. Incubate on the bench at room temperature for 3 hours. Page 4 of 9

5 C. Measurement Note: During the ELISA procedure, do not allow the wells to dry at any point. 1. Prepare Detector Solution as follows: 1 ml 20X Detector + 19 ml Dilution Buffer This is enough for a 96 well assay. If using fewer wells, adjust accordingly. Always make fresh. 2. Empty the wells and add 300 µl of Wash to each well. 3. Repeat step 2 twice more and blot the plate onto task wipers. 4. Add 200 µl of Detector Solution onto each measurement well. 5. Incubate on the bench at room temperature for 1 hour. 6. Prepare Label Solution as follows: 1 ml 20X Label + 19 ml Dilution Buffer This is enough for a 96 well assay. If using fewer wells, adjust accordingly. Always make fresh. 7. Empty the wells and add 300 µl of Wash to each well. 8. Repeat step 7 twice more and blot the plate onto task wipers. 9. Add 200 µl of Label Solution onto each measurement well. 10. Incubate on the bench at room temperature for 1 hour. 11. Prepare Development Solution as follows: 20 ml 1X Development Buffer ml of 50X Development Reagent This is enough for a 96 well assay. If using fewer wells, adjust accordingly. Always make fresh. 12. Empty the wells and add 300 µl of Wash to each well. 13. Repeat step 12 twice more and blot the plate onto task wipers. 14. Add 200 µl of Development Solution onto each measurement well. Pop any bubbles in the wells with a fine needle as rapidly as possible. 15. Set the plate in the reader. Measure at room temperature the absorbance of each well in a kinetic program at 405 nm for 30 minutes at 1 minute interval. If possible set spectrophotometer to shake plate in between measurements. Page 5 of 9

6 D. Analysis The amount of TFP enzyme captured in each well is proportional to the alkaline phosphatase activity. Therefore, the amount of TFP in a sample is equal to the change in absorbance at 405 nm/minute/micrograms of protein loaded into the well. Examine the linear rate of increase in absorbance at 405 nm as shown below. Select the start (Absorbance 2) and end points (Absorbance 1) of the linear increase and apply the following formula: 10 µg of extract from adherent cultured cells (HepG2) Background If all control and unknown samples are loaded at the high end of the linear ranges (see page 2 of this protocol), all unknown samples can be expressed as a percentage of control with the simple following formula: If higher amounts of protein are loaded onto the wells, saturation of the capturing antibody will occur in a non-linear fashion. In this situation, a standard curve must be determined with the control sample prior to determination enzyme quantity in unknown samples Page 6 of 9

7 Frequently Asked Questions 1. What is the minimum amount of cells or tissue needed to accurately measure mitochondrial TFP quantity? Although small amounts of extract are required for sample loading onto the plate, it is essential for the extract to be prepared at approximately 5.5 mg/ml. To achieve this concentration it is recommended to use a minimum of 3x10 6 cultured cells homogenized in a total volume of µl or 20 mg of tissue homogenized in 200 µl volume. 2. Is it possible to speed up this assay? Antigen-antibody reactions are dependent on many conditions such as temperature and movement of molecules. The higher the temperature and the faster the movement of molecules, the sooner the saturation of binding sites occur. This assay can be performed in about half the time if sample, detector, and label incubations are carried out at 37 C on a rotating platform. However, it is crucial to be consistent with all assays for cross-comparisons. Under the specified conditions, samples can be incubated for 1.5 hours and detector and label steps for 35 minutes each. 3. Can I use this plate to determine mitochondrial TFP quantity in tissues from rodents or other animal models? No, this kit is specific for human tissues only. Cross-reactivity against other species is not supported by this kit and may only be carried out at the own risk of the researcher. 4. Which immunogen was used to develop the antibodies used in this kit? Human Heart Mitochondria 5. What evidence do you have that the captured protein is in fact the assembled form of the mitochondrial TFP? All immunoprecipitates were analyzed by mass spectrometry for purity and completeness of the enzyme. 6. Which is the exact epitope that binds to the capture (attached to the plate) and the detector antibodies provided with this kit? The exact epitopes are unknown, but are believed to be non-linear in nature due to the fact that these antibodies target preferentially proteins in their native form. Page 7 of 9

8 FLOW CHART (For quick reference only. Be completely familiar with previous details of this document before performing the assay.) Prepare Sample (1-3 hours) Ensure sample concentration is a 5.5 mg/ml. Perform detergent extraction with 1/10 volume detergent followed by 25,000 g centrifugation for 20 min. Take supernatant. Adjust concentration to recommended dilution for plate loading. Load Plate (3 hours) Load sample(s) on plate being sure to include a normal as a positive control sample and buffer control as null reference. Incubate 3 hours at room temperature. Detector Antibody (1 hour) Empty wells and wash each well three times with 300 µl Wash. Add 200 µl of Detector Solution to every well used. Incubate for 1 hour at room temperature. Label Antibody (1 hour) Empty wells and wash each well three times with 300 µl Wash. Add 200 µl of Label Solution to every well used. Incubate for 1 hour at room temperature. Measure (0.5 hour) Empty wells, wash each well three times with 300 µl Wash. Add 200 µl of Development Solution. Measure OD 405 at 1 minute intervals for 30 minutes at room temperature. Page 8 of 9

9 MICROPLATE MS / / A B C D E F G H MS740 Page 9 of 9