ELISA for Activation of Map kinase (ERK1 and 2)
Modified from: Biosource Inc: Immunoassay Kit Catalog #KHO0091ERK1/2
To students: make a list of terms you do not understand, we will discuss them before we carry out this ELISA. Start by looking the terms up on the web, in your text.
We typically spend two weeks with this assay (testing different hormones each week)
INTRODUCTION
ERK (Extracellular Signal Regulated Kinase), a type of map kinase (MAPK; Mitogen-Activated Protein Kinase), has two closely related isoforms. ERK1, also known as MAP kinase 1 or p44 MAP kinase, is a 44 kDaprotein and ERK2, also known as MAP kinase 2 or p42 MAP kinase, is a42 kDa protein. These kinases belong to a family of serine/threoninekinases that are activated upon treatment of cells (like Xenopus oocytes) with a large variety ofstimuli including mitogens, hormones, growth factors, cytokines, and bioactive peptides (like insulin or progesterone). Cell stimulation induces the activation of a signalingcascade, the downstream effects of which have been linked to the regulation of cell growth and differentiation as well as regulation of thecytoskeleton.
ERK1 and ERK2 are serine/threonine kinases expressed broadly in normal tissues and various cell lines. They are activated through the phosphorylation of a threonine and a tyrosine residue within the activation loop by MEKs (MAPK/ERKkinases), including MEK1 (MAPK/ERK kinase 1) or MEK2. The phosphorylation moves the activation loop out of the way of the active site. Once activated, ERK1 and ERK2 can phosphorylate PXS/TPmotifs in many different proteins including cytoskeletal proteins,
translation regulators, the Rsk family of protein kinases and transcription factors, such as ELK-1.
The phosphorylation occurs on amino acids threonine 202 and tyrosine 204 of human ERK1, and on threonine 185 and tyrosine 187 of human ERK2. The way of abbreviating this is: [pTpY185/187].
The BioSource International Inc. ERK1/2 [pTpY185/187] ELISA isdesigned to detect and quantify the level of both dual-phosphorylatedERK2 at threonine 185 and tyrosine 187 (ERK2 [pTpY185/187]) and ERK1 at threonine 202 and tyrosine 204 (ERK1 [pTpY202/204]).
PRINCIPLE OF THE METHOD
The BioSource International, Inc. ERK1/2 [pTpY185/187] kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). Amonoclonal capture antibody specific for ERK1/2 (regardless of phosphorylation state) has been coated onto the wells of the microtiter strips provided. Samples, including a standard containing ERK1/2 [pTpY185/187], control specimens, and unknowns, are pipetted into these wells. Duringthe first incubation, the ERK1/2 antigens bind to the immobilized(capture) antibody.
After washing, an second antibody specific for ERK1/2 phosphorylated at threonine 185 and tyrosine 187 is added to the wells (the antibody does not bind inactive ERK without phosphate). During the second incubation, this antibody serves as a detection antibody by binding to the immobilized ERK1/2 proteins captured during the first incubation. After removal of excess detection antibody, ahorseradish peroxidase(HRP)-labeled anti-rabbit IgG (anti-rabbit IgG-HRP) is added. This THIRD antibody binds to the second detectionantibody to complete the four-member sandwich. After washing to remove all the excess antibodies, a substrate for the HRP is added, and HRP makes a blue color. The more blue, the higher the amount of active ERK1/2 [pTpY185/187] from the hormone treated oocyte.
Each little plastic well (they are connected in a strip of eight) will have the following. Note that the letter Y is used to represent antibodies (they are shaped this way):
REAGENTS PROVIDED: Note: Store all reagents at 2 - 8°C. Reagent 96 Test Kit:
ERK1/2 [pTpY185/187] Standard: Refer to vial label for quantity and reconstitution volume. This is a bit of phosphorylated ERK that is used to make sure the assay is working and to act as a standard amount.
Standard Diluent Buffer. Contains 15 mM sodium azide;25 mL per bottle. 1 bottle
ERK1/2 Antibody-Coated Wells, 96 wells per plate. 1 plate
Rabbit anti-ERK1/2 [pTpY185/187](DetectionAntibody). Contains 15 mM sodium azide; 11 mL per bottle. 1 bottle
Anti-rabbit IgG-Horseradish Peroxidase (HRP) Concentrate, (100x). Contains 3.3 mM thymol; 0.125 mL per vial.1 vial
HRP Diluent. Contains 3.3 mM thymol; 25 mL per bottle. 1 bottle
Wash Buffer Concentrate (25x); 100 mL per bottle. 1 bottle
Stabilized Chromogen, Tetramethylbenzidine (TMB); 25 mL per bottle. 1 bottle
Stop Solution; 25 mL per bottle. 1 bottle
Plate Covers, adhesive strips.
Disposal Note: This kit contains materials with small quantities ofsodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a largevolume of water to prevent azide accumulation. Avoid ingestion andcontact with eyes, skin and mucous membranes. In case of contact, rinse
affected area with plenty of water. Observe all federal, state and localregulations for disposal.
Samples should be frozen if not analyzed shortly after collection.
Avoid multiple freeze-thaw cycles of samples (WHY???). Thaw completelyand mix well prior to analysis. If particulate matter is present, centrifuge or filter prior to analysis.
Samples containing ERK1/2 [pTpY185/187] proteins extractedfrom cells should be diluted with Standard Diluent Buffer at least1:10. This dilution is necessary to reduce the matrix effect of the cell lysis buffer. When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells. Cover or cap all reagents when not in use. Read absorbances within 2 hours of assay completion. In-house controls should be run with every assay. If control valuesfall outside pre-established ranges, the accuracy of the assay issuspect. All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefullyon absorbent paper. Never insert absorbent paper directly into the
wells. Because Stabilized Chromogen is light sensitive, avoid prolongedexposure to light. Also avoid contact between StabilizedChromogen and metal, or color may develop.
FOR THE FIRST LAB MEETING TIME: PROCEDURE FOR EXTRACTION OF PROTEINS FROMCELLS
Oocyte collection:
The ovaries from Xenopus females are surgically removed and placed into room temperature 1X O-R2 (oocyte-ringer’s) buffer solution (83 mM NaCl, 0.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, ph 7.5). Healthy large (stage VI) oocytes are then manually isolated from the follicular membranes using forceps.
1. Using a plastic transfer pipette whose end has been cut ~ 4 mm from the end to increase the diameter of the opening, transfer groups of 10 oocytes to large plastic Petri dishes containing 30 ml 1X OR-2(by widening the tip of the pipette you minimize mechanical damage that may occur during transfer).
Some student groups will collect control cell data; no treatment with insulin.
2. Using the appropriate size Pipetman, add the appropriate amount of insulin to each experimental group to give a final concentration of 1 or 2 µM (Formula for calculation: M1V1=M2V2 where M1= final concentration, V1= 1 ml, M2 = stock concentration and . V2= volume of stock to be added). Show your calculations in your lab notebook. DO NOT ADD INSULIN DIRECTLY ON TOP OF THE CELLS, BUT TO THE SIDE and then gently swirl. WHY? Insulin is in HCl- do not add acid directly to cells; and do not add the concentrated solution of insulin directly to cells because the concentration bolus would be too high!
3. Following insulin, metformin or progesterone addition, at appropriate times, homogenize the oocytes. ADD 10 OOCYTES TO THE BOTTOM OF A V VIAL (also called a 1.7 ml Eppendorf v-vial) AT ROOM TEMP, THEN REMOVE EXCESS SOLUTION. THEN ADD 200 uL of ICE COLD HOMOGENIZATION SOLUTION to the v vial and cells—IMMEDIATELY homogenize with the blue pestle (do not let the oocytes sit in the cold solution as this will alter them). Slowly twist the pestle as you come down on each stroke to ensure that every cell is homogenized.
The homogenization buffer is used to prevent any destruction of map kinase, or dephosphorylation or new phosphorylation of map kinase due to the simple crushing and homogenizing of the cell. We lower the temperature of the solution to turn off all enzymes, and there are multiple enzyme inhibitors in the homogenation buffer.
We will go through the following list and explain their purpose…
Cell Homogenization Buffer:
- 10 mM Tris, pH 7.4
- 100 mM NaCl
- 1 mM EDTA
- 1 mM EGTA
- 1 mM NaF
- 20 mM Na4P2O7
- 2 mM Na3VO4
- 1% Triton X-100
- 10% glycerol
- 0.1% to 1.0% SDS (See section C below)
- 0.5% deoxycholate
- 1 mM PMSF (stock is 0.3 M in DMSO)
- Protease inhibitor cocktail (ex., Sigma Cat. # P-2714) (reconstituted
according to manufacturer’s guideline). Add 250 µL per 5 mL Cell
Extraction Buffer. This buffer is stable for 2-3 weeks at 4C or for up to 6 months when
aliquoted (without protease inhibitors and PMSF added) and stored at -20 C. When stored frozen, the Cell Extraction Buffer should be thawed on ice. Important: add the protease inhibitors just before using. The stability of protease inhibitor supplemented Cell Extraction Buffer is 24 hours at 4C. PMSF is very unstable and must be added prior to use, even if added previously.
4. Incubate the homogenate on ice for 5-10 min. this extracts all ERK from membranes, and other sites; we want all ERK floating freely in the solution.
5. Centrifuge the samples at 10000 revolutions per minute for 10 min using the microfuge (hopefully, in the 4°C cooler to keep samples cold, enzymes inactive). This gets rid of cellular crud; leaving the ERK in the supernatant.
6. Transfer the supernatant (aqueous layer) for each sample to clean v-vials using a Pipetman 200 and store at -20°Cor -80 C freezer—see figure below- note hold the v vial so the pellet is on top, suck from underneath it to remove supernatant. These samples areready for assay. Avoid multiplefreeze/thaw cycles.End of Day 1.
For the SECOND LAB MEETING TIME:
(note that I should talk only after first incubation is started).
REAGENT PREPARATION AND STORAGE
We typically do this for students: Storage and Final Dilution of Anti-rabbit IgG Horseradish Peroxidase (HRP) Please Note: The Anti-rabbit IgG-HRP 100x concentrate is in 50%glycerol. This solution is viscous. To ensure accurate dilution, allowAnti-rabbit IgG-HRP concentrate to reach room temperature. Gentlymix. Pipette Anti-rabbit IgG-HRP concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with cleanabsorbent paper.
Dilute 10 µL of this 100x concentrated solution with 1 mL of HRPDiluent for each 8-well strip used in the assay. Typically, we need two strips, so this would be 20uL of stock with yellow cap plus 2ml of HRP diluent (green cap)- put into a 1.8 ml v vial. Label as Anti-rabbitIgG-HRP Working Solution (we will do this for you and provide the working HRP Diluent).
Dilution of Wash Buffer (students do)
Allow the 25x concentrate to reach room temperature and mix to ensure that any precipitated salts have redissolved. Dilute 1 volume of the 25xWash Buffer Concentrate with 24 volumes of deionized water (e.g., 50 mL may be diluted up to 1.25 liters, 100 mL may be diluted up to 2.5 liters). Label as WorkingWash Buffer. Store both the concentrate and the Working Wash Buffer in the refrigerator. The diluted buffer should be used within 14 days.
ASSAY METHOD: PROCEDURE AND CALCULATIONS
Be sure to read the Procedural Notes/Lab Quality Control section
before carrying out the assay.
Allow all reagents to reach room temperature before use. Gently mix allliquid reagents prior to use. We will use a plate reader to record the optical density for different samples located in different small wells. A plate reader is just a spectrophotometer that is set up to rapidly read many small samples (it also can be set to vibrate the rack of plastic wells as it is difficult to mix the small amount of solution in the wells)- remember we determined the concentration of insulin in a cuvette in the spectrophotometer and we used a relatively large volume (half a ml or more). Our DynaTech MR5000 plate reader is below- note the control panel on the lower left and the samples go on the black platform in the upper right (each sample is in a small well and the wells are held together in a frame or rack):
1.Determine the number of 8-well strips needed for the assay. Insertthese in the frame(s) for current use. (Re-bag extra strips and frame.Store these in the refrigerator for future use.)
2.Add 100 µL of the Standard Diluent Buffer (this solution is already made)to well A1 that will be used for as a blank –these well or wells will have no ERK in them and this value is typically subtracted from all other values. However, the blank value we get is way too high- why?
3.Add 84 µl Standard Diluent Buffer (1:6.25 dilution) to all wells. Then add 16 µl of cellular homogenate to the other wells; make a chart of what sample goes where (well A2 is control 1, well A3 is control 2, etc). Tap gently on side of plate tothoroughly mix—better yet program the plate reader to vibrate (Program #1, no name, hit escape to start). We dilute samples to reduce the final SDS concentration to below 0.01% (SDS detergent interferes with the assay, so it has to be very dilute).
4.Cover wells with plate cover and incubate for 45min to 1 hour at roomtemperature. Unless told differently, take this opportunity to review this and other procedures and ask any questions you might have. Exam is mid-March. Fill out your lab notebook.
5. To remove solution in the wells, flick out the solution in the wells and then pound the rack upside (gently) on absorbent paper towels. This gets rid of all protein except the active phosphor-ERK that is stuck to the bottom of the wells with the capture antibody. BE VERY CAREFUL NOT TO SCRATCH OR TOUCH THE BOTTOM OF THE WELLS.
6. Wash wells 3 times. DIRECTIONS FOR WASHING: add ____ mL of 25X wash stock solution to 100 ml graduated cylinder, then top off with deionized water to 100 ml. (for making the wash solution, see procedure above).
Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided. Fill the wells with at least 0.4 mL of diluted wash solution (use the multipipetman). Let soak for 15 to 30 seconds, then remove the liquid. Repeat.
After the washing procedure, the plate is inverted and tapped dry on absorbent tissue.
7. Using the 200uL Pipetman, pipette 100 µL of anti-ERK1/2 [pTpY185/187] (DetectionAntibody) solution into each well. Tap gently on the side of the plate to mix.
7. Cover wells with plate cover and incubate for 0.5hour at roomtemperature.
8. Thoroughly remove or decant solution from wells and discard the liquid. Wash wells 4 times. See the DIRECTIONS FOR WASHING above.
9. Add 100 µL anti-rabbit IgG-HRP Working Solution to each well. This solution was prepared already.
10. Cover wells with the plate cover and incubate for 30 minutes atroom temperature.
11. Thoroughly remove or decant solution from wells and discard the liquid. Wash wells 4 times. See DIRECTIONS FOR WASHING above.
12. Add 100 µL of Stabilized Chromogen (this is the substrate for the HRP) to each well (at 15s intervals). The liquid inthe wells will begin to turn blue.
13. Incubate for 30 minutes at room temperature and in the dark.
Please Note: Do not cover the plate with aluminum foil ormetalized mylar.
14. Add 100 µL of Stop Solution to each well (at 15s intervals)- this stops color development. Tap side of plate gentlyto mix. The solution in the wells should change from blue to yellow.
15. Read the absorbance of each well at 450 nm (read the plate within2 hours after adding the Stop Solutionor color may fade).
Instructions for plate reader (look at the green strip at the top for command words):
-select “Manual”
-select “Single” wavelength
-set test filter to 450nM (hit “next”)
-select “Enter”
-use directional arrows to read absorbance of each well
The control panel for the plate reader is enlarged below:
16. You will be given data for a a plot of phosphoERK standard amount versus the OD at 450nm. This plot can be used to determine the actual concentration of ERK in your samples. You use a regression to find the standard line, and then put in your OD 450 and find the concentration of ERK.
What is the amount of ERK1/2 [pTpY185/187] in your sample? The dilution factor for your oocyte sample is 6.25 (16 uL into 100 ul total; so 100/16= 6.25).
Then, remember that this is the amount from 10 cells, so divide by 10 to get the amount of active phosphoERK (in units per oocyte).
LIMITATIONS OF THE PROCEDURE
Do not extrapolate the standard curve beyond the 100 Units/mLstandard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute samples >100 Units/mL with Standard Diluent Buffer; re-analyze these and multiply results by theappropriate dilution factor. The influence of various extraction buffers has not been thoroughlyinvestigated. The rate of degradation of native ERK1/2 or dephosphorylation of ERK1/2 [pTpY185/187] in various matrices hasnot been investigated. Although ERK1/2 degradation ordephosphorylation of ERK1/2 [pTpY185/187] in the Cell Extraction Buffer described in this protocol has not been seen to date, thepossibility of this occurrence cannot be excluded.
PERFORMANCE CHARACTERISTICS
SENSITIVITY
The analytical sensitivity of this assay is <0.8 Units/mL of humanERK1/2 [pTpY185/187]. This was determined by adding two standarddeviations to the mean O.D. obtained when the zero standard wasassayed 30 times.
The sensitivity of this ELISA was compared to Western blotting usingknown quantities of ERK1/2 [pTpY185/187]. The data presented in Figure 3 show that the sensitivity of the ELISA is approximately 4xgreater than that of Western blotting.
Make sure that you view the Western blotting video so you know about this other procedure that quantifies protein.
REFERENCES on Map Kinase (ERK)
1.Cobb, M.H., et al. (1994) The mitogen-activated protein kinases,
ERK1 and ERK2. Semin. Cancer Biol. 5(4):261-268.