Elisa Kit
Elisa Kit
Elisa Kit
FOR LABORATORY RESEARCH USE ONLY! NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH
ENTIRE PROCEDURE BEFORE BEGINNING! THIS IS JUST A REFERENCE PROTOCOL! PLEASE USE THE MANUAL FOUND IN
YOUR ELISA KIT FOR EXACT INFORMATION!
INTENDED USE
This BG AMPK ELISA kit is intended for laboratory research use only and not for use in diagnostic or therapeutic procedures.
The stop solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a
spectrophotometer. In order to measure the concentration of AMPK in the sample, this AMPK ELISA Kit includes a set of
calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a
standard curve of Optical Density versus AMPK concentration. The concentration of in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
PRINCIPLE OF THE ASSAY
The coated well immunoenzymatic assay for the quantitative measurement of AMPK utilizes a multiclonal anti-AMPK antibody
and an AMPK-HRP conjugate. The assay sample and buffer are incubated together with AMPK-HRP conjugate in pre-coated
plate for one hour. After the incubation period, the wells are decanted and washed five times. The wells are then incubated with a
substrate for HRP enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stop solution is
added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at
450nm in a microplate reader. The intensity of the color is inversely proportional to the AMPK concentration since AMPK from
samples and AMPK-HRP conjugate compete for the anti-AMPK antibody binding site. Since the number of sites is limited, as
more sites are occupied by AMPK from the sample, fewer sites are left to bind AMPK-HRP conjugate. Standards of known
AMPK concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of
the color (Optical Density) to the concentration of AMPK. The AMPK concentration in each sample is interpolated from this
standard curve.
MATERIALS
All reagents provided are stored at 2-8 C. Refer to the expiration date on the label.
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MICROTITER PLATE
ENZYME CONJUGATE
STANDARD.1
STANDARD.2
STANDARD.3
STANDARD.4
STANDARD.5
STANDARD.6
SUBSTRATE A
SUBSTRATE B
STOP SOLUTION
WASH SOLUTION (x100)
INSTRUCTION
LYSIS BUFFER SOLUTION
96 wells
6.0 mL
0 ng/mL
5 ng/mL
10 ng/mL
25 ng/mL
50 ng/mL
100 ng/mL
6.0 mL
6.0 mL
6.0 mL
10 mL
1
6.0 mL
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
NOTE: The LYSIS BUFFER SOLUTION is used only when the sample is cell culture fluid & body fluid & tissue homogenate;
if the sample is serum or blood plasma, then the LYSIS BUFFER SOLUTION is a superfluous reagent.
The kinds of sample:
sample: serum or blood plasma
sample: cell culture fluid & body fluid & tissue homogenate
SAMPLE COLLECTION AND STORAGE
Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before a centrifugation for 15minutes at
approximately 1000 x g. Remove serum and perform the assay immediately or aliquot and store samples at -20 C or -80C.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2-8C
within 30minutes of collection. Store samples at -20C or -80C.Avoid repeated freeze-thaw cycles.
Cell culture fluid and other biological fluids-Remove particulates by centrifugation and assay immediately or aliquot and store
samples at -20C or -80C. Avoid repeated freeze-thaw cycles.
NOTE: Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2-8C, otherwise samples must
be stored at -20C(2months) or -80C(6months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles.
When performing the assay, warm up samples to room temperature slowly. DO NOT USE HEAT-TREATED SAMPLES.
SAMPLE PREPARATION
1. BLUEGENE (BG) is only responsible for the kit itself, but not for the samples consumed during the assay. The user should
calculate the possible amount of the samples used in the whole test. Please reserve sufficient amount of samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must
determine the optimal sample dilutions for their particular experiments.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
4. Owing to the possibility of mismatching between antigen from other resource and antibody used in our kits (e.g., antibody
targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not
be recognized by our products.
5. Influenced by the factors including cell viability, cell number and also sampling time, samples from cell culture supernatant
may not be detected by the kit.
6. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may
occur in those samples and finally lead to wrong results.
REAGENTS PREPARATION
1. Bring all kit components and samples to room temperature (18-25 ) before use.
2. Dispense 10 L of LYSIS BUFFER SOLUTION into 100 L specimens, mix and stand for one hour (The proportion of LYSIS
BUFFER and Specimens should be no less than 1:10). (NOTE: This step is required when the sample is cell culture fluid & body
fluid & tissue homogenate; if the sample is serum or blood plasma, then this step should be skipped.)
3. Wash Solution - Dilute 10 mL of Wash Solution concentrate (100) with 990 mL of deionized or distilled water to prepare
1000 mL of Wash Solution (1).
ASSAY PROCEDURE
Prepare all Standards before starting assay procedure (Please read Reagents Preparation). It is recommended that all Standards
and Samples be added in duplicate to the Microtiter Plate.
1. Secure the desired numbers of coated wells in the holder then add 100 L of Standards or Samples to the appropriate well of the
antibody pre-coated Microtiter Plate.
2. Add 50 L of Conjugate to each well. Mix well. Mixing well in this step is important. Cover and incubate the plate for 1 hour
at 37C.
3. Wash the Microtiter Plate using one of the specified methods indicated below:
3.1 Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Fill in
each well completely with diluted wash solution, and then aspirate contents of the plate into a sink or proper waste container.
Repeat this procedure five times for a total of FIVE washes. After washing, invert plate, and blot dry by hitting the plate onto
absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the
plate to assure that all strips remain securely in frame. Complete removal of liquid at each step is essential to good
performance.
3.2 Automated Washing: Wash plate FIVE times with diluted wash solution (350-400 L/well/wash) using an autowasher. After
washing, dry the plate as above. It is recommended that the washer be set for a soaking time of 10 seconds and shaking time
of 5 seconds between each wash.
4. Add 50 L Substrate A and 50 L Substrate B to each well, subsequently. Cover and incubate for 10 minutes at 20-25C.
(Avoid sunlight).
5. Add 50 L of Stop Solution to each well. Mix well.
6. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader immediately.
BG CALCULATION OF RESULTS
1. This standard curve is used to determine the amount of an unknown sample. Construct a standard curve by plotting the average
O.D. (450 nm) for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis, and draw a best fit
curve through the points on the graph.
2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the
blank control before result interpretation. Construct the standard curve using graph paper or statistical software.
3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard
curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in
result. Each user should obtain their own standard curve.
5. Standard curve:
2.5
2.0
1.5
C1
C5
1.0
C9
0.5
0.0
0
10
Concentrations