ab140358 AMPK alpha 1 ELISA kit Instructions for Use For the quantitative measurement of AMPK alpha 1 concentrations in human, mouse and rat cell culture and tissue lysates. This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Introduction 3 2. Assay Summary 6 3. Kit Contents 7 4. Storage and Handling 7 5. Additional Materials Required 8 6. Preparation of Reagents 9 7. Sample Preparation 11 8. Assay Procedure 13 9. Data Analysis 15 10. Specificity 18 11. Cross-reactivity 19 12. Troubleshooting 20 2
1. Introduction Principle: ab140358 5'-AMP-activated protein kinase catalytic subunit alpha-1 (AMPKα1, PRKAA1) ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in-vitro enzyme-linked immunosorbent assay for the accurate quantitative measurement of AMPK α1 subunit in human, rat and mouse lysates. The assay employs an AMPKα1 specific mouse monoclonal antibody bound to the plate. Standards and samples are pipetted into the wells in conjunction with a rabbit monoclonal primary detector antibody. After washing away excess protein and unbound detector antibody, an HRP-conjugated secondary detector antibody (HRP Label) specific for the primary detector antibody is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of AMPKα1 bound. The developing blue color is measured at 600 nm. Optionally, the reaction can be stopped by adding hydrochloric acid which changes the color from blue to yellow and the intensity can be measured at 450 nm. 3
Background: AMP-activated protein kinase (AMPK) is an energy sensor protein kinase that plays a key role in regulating cellular energy homeostasis. Mammalian AMPK is a heterotrimer kinase, containing a catalytic subunit (α) and two regulatory subunits (β and γ). Each subunit has different isoforms (α1, α2, β1, β2, γ1, γ2, γ3) with differential tissue expression, cellular localization and functionality. Human AMPK has a 98.7% sequence similarity with mouse AMPK and a 99% sequence similarity with rat AMPK. It has been hypothesized that when ADP or AMP are present at high levels, these nucleotides bind directly to the γ subunit, leading to a conformational change that allows phosphorylation of Thr172 at the α subunit. Phosphorylation of AMPK α activates the kinase, which leads to downstream effects concerted to increase catabolic pathway and suppress anabolic pathways in order to restore levels of cellular ATP and ultimately control cell fate. AMPK is activated physiologically due to stresses such as low nutrients and prolonged exercise. Furthermore AMPK may be activated pharmacologically by metformin (the most widely prescribed Type 2 diabetes drug), phenformin, AICAR and resveratrol. Activation of AMPK due to low nutrients leads to suppression of the mammalian target of rapamycin complex 1 (mtorc1) pathway allowing coordination and control of cell growth and autophagy. Furthermore, AMPK also controls metabolism via direct phosphorylation of metabolic enzymes such as Acetyl-CoA carboxylase (ACC1 and ACC2) HMG-CoA reductase, hormone 4
sensitive lipase (HSL), adipose triglyceride lipase (ATGL), Insulin receptor substrate 1 (IRS1) and phosphofructo-kinase (PFKFB). Control of metabolism also occurs long term through control of transcription and chromatin structure via phosphorylation of transcription factors (SREBP1, PPARγ), co-activators (CRTC family, PGC1α), acetyltransferase p300, histone H2B and histone deacetylases (HDACs class IIa). Activation of AMPK has also been linked to circadian clock regulation via phosphorylation of Cry1, coupling daily light and dark cycles to the metabolic control of fed and fasting cycles. In addition, it has been suggested that AMPK may control cell polarity and cytoskeletal dynamics in some settings. 5
2. Assay Summary Equilibrate all reagents to room temperature. Prepare all the reagents, samples, and standards as instructed. Add 25 µl of 2X standard or 2X sample to each well used and overlay with 25 µl of detector antibody. Incubate 3 hours at room temperature. Aspirate and wash each well three times. Add 50 μl of prepared HRP labeled secondary detector antibody. Incubate 1 hour at room temperature. Aspirate and wash each well four times. Add 100 μl TMB Development Solution to each well. Immediately begin recording the color development with elapsed time at 600 nm for 15 minutes. Alternatively add a Stop solution at a user-defined time and read at 450 nm. 6
3. Kit Contents Item 20X Buffer Extraction Buffer 10X Blocking Buffer TMB Development Solution Quantity 25 ml 15 ml 6 ml 12 ml 20X AMPK α1 Detector Antibody 250 µl 10X HRP Label Human AMPK α1 Standard (1 µg) AMPK α1 Microplate (12 x 8 antibody coated well strips) 1 ml 1 vial 96 Wells 4. Storage and Handling Store all components at 4 C (do not freeze the lyophilized protein). This kit is stable for 6 months from receipt. After reconstitution the standard should be aliquoted and stored at -80 C. Unused microplate strips should be returned to the pouch containing the desiccant and resealed. 7
5. Additional Materials Required Microplate reader capable of measuring absorbance at 600 nm (or 450 nm after addition of Stop solution - not supplied. Method for determining protein concentration (BCA assay recommended). Deionized water Multi- and single-channel pipettes PBS (1.4 mm KH 2 PO4, 8 mm Na 2 HPO 4, 140 mm NaCl, 2.7 mm KCl, ph 7.3) Tubes for standard dilution Stop solution (optional) 1N hydrochloric acid Optional plate shaker for all incubation steps Well plate cover or seals 8
6. Preparation of Reagents 6.1. Equilibrate all reagents and samples to room temperature (18-25 C) prior to use. 6.2. Prepare 1X Wash Buffer by adding 20 ml of 20X Buffer to 380 ml of nanopure water and mix thoroughly. 6.3. Prepare 1X Incubation Buffer by adding 6 ml of 10X Blocking Buffer to 54 ml of 1X Wash Buffer and mix thoroughly. Excess unused 1X Incubation buffer may be stored at -20 C for 6 months after performing the ELISA. 6.4. Prepare the 2X AMPK α1 Detector Antibody by diluting the 20X AMPK α1 Detector Antibody 10-fold with 1X Incubation Buffer immediately prior to use. Prepare 250 µl for each 8 well strip used. 6.5. Prepare the 1X HRP Label (secondary antibody) by diluting the 10X HRP Label 1:10 with 1X Incubation Buffer immediately before use. Prepare 500 µl for each 8 well strip used. 6.6. Reconstitute the 1 µg Human AMPK α1 Standard by adding 200 µl of nanopure water and vortex vigorously; the final concentration will be 5 µg/ml. Hold for 10 minutes on ice and repeat vortex. Aliquot and freeze spare tubes at -80 C (avoid freeze/thaw cycles). 9
6.7. Prepare serially diluted Human AMPK α1 Standards 6.7.1. First label tubes #1-7. Add 75 μl of 1X Incubation Buffer to each of tubes #2 through #7. 6.7.2. Prepare the top standard curve sample (tube #1) at 2,000 ng/ml by diluting 60 µl of the 5 µg/ml reconstituted protein into 90 μl of 1X Incubation Buffer. 6.7.3. Transfer 75 μl from tube #1 to tube #2 and mix thoroughly. 6.7.4. With a fresh pipette tip transfer 75 μl from #2 to #3 and mix thoroughly. 6.7.5. Repeat for Tubes #4 through #7. 6.7.6. Use 1X Incubation buffer as the zero standard tube labeled #8. 6.7.7. Prepare fresh standards for each assay. 75 l 75 l 75 l 75 l 75 l 75 l 2 3 4 5 6 7 2000 ng/ml 1/2 1/4 1/8 1/16 1/32 1/64 in 1X Incubation buffer 10
7. Sample Preparation Note: The extraction buffer provided with this kit may be supplemented with phosphatase inhibitors, PMSF and protease inhibitor cocktail prior to use. Supplements should be used according to manufacturer s instructions. 7.1 Preparation of extract from cell pellets (Human, mouse, rat). 7.1.1 Collect non-adherent cells by centrifugation or scrape to collect adherent cells from the culture flask. Typical centrifugation conditions for cells are 500 x g for 10 min at 4 C. 7.1.2 Rinse cells twice with PBS. 7.1.3 Solubilize cell pellet at 4 x 10 7 /ml in Extraction Buffer. 7.1.4 Incubate on ice for 20 minutes. Centrifuge at 15,000 x g for 10 minutes at 4 C. Transfer the supernatants into clean tubes and discard the pellets. Assay samples immediately or aliquot and store at -80 C (avoid freeze/thaw cycles). The sample protein concentration in the extract may be quantified using a protein assay. 11
7.2 Preparation of extracts from tissue homogenates (Human tissues only, not recommended for mouse). 7.2.1 Tissue lysates are typically prepared by homogenization of tissue that is first minced and thoroughly rinsed in PBS to remove blood (dounce homogenizer recommended). 7.2.2 Homogenize 100 to 200mg of wet tissue in 500µL to 1 ml of the supplied extraction buffer. For lower amounts of tissue adjust volumes accordingly. 7.2.3 Incubate on ice for 20 minutes. Centrifuge at 10,000 xg, for 10 minutes at 4 C. Transfer the supernatants into clean tubes and discard the pellets. Assay samples immediately or aliquot and store at -80 C (Avoid freeze/thaw cycles). The sample protein concentration in the extract may be quantified using a protein assay. 12
8. Assay Procedure Equilibrate all reagents to room temperature prior to use. It is recommended all samples and standards be assayed in duplicate. If available use a plate shaker for all incubation steps at 300rpm. 8.1 Prepare all reagents, working standards, and samples as directed in the previous sections. 8.2 Remove unused microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal. 8.3 Add 25 µl of each serially diluted Human AMPK α1 Standards or test sample per well. Also include a 1X Incubation Buffer as a zero standard. 8.4 The samples should be diluted at 2X the final desired concentration within the working range of the assay in 1X Incubation Buffer. As a guide, typical ranges of sample concentration are shown below in Data Analysis. Add 25 µl of each sample at 2X the final desired concentration to wells. 8.5 Overlay 25 µl 2X AMPK α1 Detector Antibody (from step 6.4) to the serially diluted standard or sample. Cover/seal the plate and incubate for 3 hours at room temperature. 13
8.6 Aspirate each well and wash, repeat this twice more for a total of three washes. Wash by aspirating or decanting from wells then dispensing 300 µl 1X Wash Buffer into each well as described above. Complete removal of liquid at each step is essential to good performance. After the last wash, remove the remaining buffer by aspiration or decanting. Invert the plate and blot it against clean paper towels to remove excess liquid. 8.7 Add 50 µl 1X HRP Label (from step 6.5) to each well used. Cover/seal the plate and incubate for 1 hour at room temperature. 8.8 Repeat the aspirate/wash procedure above. 8.9 Add 100 µl TMB Development Solution to each empty well and immediately record the blue color development with time in the microplate reader prepared with the following settings: Mode: Wavelength: Time: Interval: Shaking: Kinetic 600 nm up to 15 min. 20 sec. - 1 min. Shake between readings Alternative In place of a kinetic reading, at a user defined time, record the endpoint OD data at (i) 600 nm or (ii) stop the reaction by adding 100 µl stop solution (1N HCl) to each well and record the OD at 450 nm. Analyze the data as described below. 14
mod/min (600nm) 9. Data Analysis Average the duplicate standard readings and plot against their concentrations after subtracting the zero standard reading. Draw the best smooth curve through these points to construct a standard curve. Most plate reader software or graphing software can plot these values and curve fit. A four parameter algorithm (4PL) usually provides the best fit, though other equations can be examined to see which provides the most accurate results (e.g. linear, semi-log, log/log, 4 parameter logistic). Read AMPK α1 protein concentrations for unknown and control samples from the standard curve plotted. Samples producing signals greater than that of the highest standard should be further diluted in 1X Incubation Buffer and reanalyzed, then multiplying the concentration found by the appropriate dilution factor. TYPICAL STANDARD CURVE - For demonstration only. 100 10 1 10 100 1000 AMPK 1 [ng/ml] Figure 1. Example of standard curve 15
mod/min (600nm) TYPICAL SAMPLE RANGE 100 10 1 0.1 10 100 1000 Hek293T extract [ g/ml] Figure 2. Example of range in Hek293T cells TYPICAL SAMPLE RANGE Sample Type AMPK α1 standard (Human recombinant protein) Hek293T C2C12 H4IIE Typical working ranges Range 1.4 1000 ng/ml 10 500 µg/ml 60 1000 µg/ml 60 1000 µg/ml SENSITIVITY Calculated minimum detectable dose = 1.4 ng/ml (zero dose n=14 standard deviations) 16
LINEARITY OF DILUTION Hek293T dilution [500 µg/ml] % Expected Value 1:1 100% 1:2 99.4% 1:4 92.5% 1:8 70% RECOVERY Sample Type Average Recovery (%) Range (%) 50% Culture media (8 dilutions) 107% 79 124% 10% Serum (8 dilutions) 77% 64 95% 50% Extraction buffer 90% 75 107% REPRODUCIBILITY Parameter CV% Intra (n= 18) 5.6% Inter (n= 3) 10% 17
10. Specificity Marker Human AMPK α1 standard C2C12 myotubes C2C12 myoblasts Starved C2C12 myoblasts H4IIE Starved H4IIE Hek293T Starved Hek293T Figure 3. Western blot using capture antibody anti-ampk α1. Endogenous levels of AMPK α were measured from various cell lysates on western blot using the capture antibody provided in the kit. Samples were loaded as follows: (1) Marker, (2) AMPK α1 standard protein (tagged) 40 ng, (3) mouse C2C12 myotubes 40 µg (4) C2C12 myoblasts in 10F media 40 µg, (5) C2C12 myoblasts in 0F media 40 µg, (6) rat H4IIE in 10F media 40 µg, (7) H4IIE in 0F media 40 µg, (8) human Hek293T in 10F media 40 µg, (9) Hek293T in 0F media 40 µg. Blot was performed under reducing conditions. Membranes were blocked with 5% Milk in TBS + 0.1% Tween-20 (TBST). Primary antibody was incubated in 1% Milk in TBST overnight at 1/1000. Secondary antibody (GAM HRP) was incubated in 1X blocking buffer 18
mod/min [600nm] mod/min [600nm] 11. Cross-reactivity 100 100 10 10 1 1 0.1 10 100 1000 H4IIE Lysate [ g/ml] 0.1 10 100 1000 C2C12 Lysate [ g/ml] Figure 4. AMPK α1 ELISA assay - dynamic range in rodent cell lines. Relative levels of AMPK α1 can be measured in rodent samples. Left panel shows mod/min data obtained with H4IIE (rat hepatocarcinoma) cells loaded in a range from 60 to 1000 µg/ml. Right panel shows same type of data on C2C12 (mouse myoblasts). 19
12. Troubleshooting Problem Cause Solution Inaccurate Pipetting Check pipettes Poor standard curve Improper standard dilution Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing Low Signal Large CV Low sensitivity Incubation times too brief Inadequate reagent volumes or improper dilution Plate is insufficiently washed Contaminated wash buffer Improper storage of the ELISA kit Ensure sufficient incubation times; change to overnight standard/sample incubation Check pipettes and ensure correct preparation Review manual for proper wash technique. If using a plate washer, check all ports for obstructions Prepare fresh wash buffer Store the reconstituted protein at -80 C, all other assay components at 4 C. Keep substrate solution protected from light. 20
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