Rat Ultra Sensitive Heat Shock
Protein 70, HSP-70
ELISA Kit
(96 T)
This immunoassay kit allows for the in vitro quantitative determination of rat
HSP-70 concentrations in serum, plasma and other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an antibody specific to HSP-70. Standards or samples are then
added to the appropriate microtiter plate wells with a
biotin-conjugated antibody preparation specific for HSP-70 and
Avidin conjugated to Horseradish Peroxidase (HRP) is added to
each microplate well and promote. Then a TMB (3,3,5,5
tetramethyl-benzidine) substrate solution is added to each well.
Only those wells that contain HSP-70, biotin-conjugated antibody
and enzyme-conjugated Avidin will exhibit a change in color. The
enzyme-substrate reaction is terminated by the addition of a
sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm ± 2 nm. The
concentration of HSP-70 in the samples is then determined by
comparing the OD of the samples to the standard curve.
DETECTION RANGE
39 pg / ml-2500 pg / ml. The standard curve concentrations used
for the ELISA's were 2500 pg / ml, 1250 pg / ml, 625 pg / ml, 312
pg / ml, 156 pg / ml, 78 pg / ml, 39 pg / ml.
SPECIFICITY
This assay recognizes recombinant and natural rat HSP-70. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat HSP-70 is typically less
than 9.8 pg / ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD)
was defined as the lowest protein concentration that could be
differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
Wash Buffer
1 x 20 ml
(25 × concentrate)
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
STORAGE
1. Unopened test kits should be stored at 2-8 ° C upon receipt
and the microtiter plate should be kept in a sealed bag. The
test kit may be used throughout the expiration date of the kit,
provided it is stored as prescribed above. Refer to the
package label for the expiration date.
2. Opened test plate should be stored at 2-8 ° C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
kits will remain stable until the expiring date shown, provided it
is stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less
and an optical density range of 0-3 OD or greater at 450nm
wavelength is acceptable for use in absorbance
measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate,
warm up to room temperature and mix gently until the
crystals have completely dissolved. Dilute 20 ml of Wash
Buffer Concentrate into deionized or distilled water to prepare
500 ml of Wash Buffer.
2. Standard Centrifuge the standard vial at 6000-10000rpm
for 30s. Reconstitute the Standard with 1.0 ml of Sample
Diluent. This reconstitution produces a stock solution of 2500
pg / ml. Allow the standard to sit for a minimum of 15 minutes
with gentle agitation prior to making serial dilutions. The
undiluted standard serves as the high standard (2500 pg / ml).
The Sample Diluent serves as the zero standard (0 pg / ml).
Prepare fresh for each assay. Use within 4 hours and discard
after use.
3. Biotin-antibody Centrifuge the vial before opening. Dilute
to the working concentration using Biotin-antibody
Diluent (1: 100), respectively.
4. HRP-avidin Centrifuge the vial before opening. Dilute to the
working concentration using HRP-avidin Diluent (1: 100),
respectively.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate
washer.
An incubator which can provide stable incubation conditions
up to 37 ° C ± 0.5 ° C.
SAMPLE COLLECTION AND STORAGE
Serum Use a serum separator tube (SST) and allow
samples to clot for 30 minutes before centrifugation for 15
minutes at 1000 g. Remove serum and assay immediately or
aliquot and store samples at -20 ° C. Centrifuge the sample
again after thawing before the assay. Avoid repeated
freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
30 minutes of collection. Assay immediately or aliquot and
store samples at -20 ° C. Centrifuge the sample again after
thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
All the reagents should be added directly to the liquid level in the well. The
pipette should avoid contacting the inner wall of the well.
1. Serum and plasma samples require a 20-fold dilution into
Sample Diluent. The suggested 20-fold dilution can be
achieved by adding 15μl sample to 285μl of Sample Diluent.
2. Add 100μl of Standard, Blank, or Sample per well. Cover with
the adhesive strip. Incubate for 2 hours at 37 ° C.
3. Remove the liquid of each well, don't wash.
4. Add 100μl of Biotin-antibody working solution to each well.
Incubate for 1 hour at 37 ° C. Biotin-antibody working
solution may appear cloudy. Warm up to room temperature
and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three
times for a total of three washes. Wash: Fill each well with
Wash Buffer (200μl) and let it stand for 2 minutes, then
remove the liquid by flicking the plate over a sink. The
remaining drops are removed by patting the plate on a paper
towel. Complete removal of liquid at each step is essential to
good performance.
6. Add 100μl of HRP-avidin working solution to each well.
Cover the microtiter plate with a new adhesive strip. Incubate
for 1 hour at 37 ° C.
7. Repeat the aspiration and wash five times as step 4.
8. Add 90μl of TMB Substrate to each well. Incubate for 10-30
minutes at 37 ° C. Keeping the plate away from drafts and
other temperature fluctuations in the dark.
9. Add 50μl of Stop Solution to each well when the first four
wells containing the highest concentration of standards
develop obvious blue color. If color change does not appear
uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard, control, and
sample and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer
software capable of generating a four parameter logistic (4-PL)
curve-fit. As an alternative, construct a standard curve by plotting
the mean absorbance for each standard on the y-axis against
the concentration on the x-axis and draw a best fit curve through
the points on the graph. The data may be linearized by plotting
the log of the HSP-70 concentrations versus the log of the OD
and the best fit line can be determined by regression analysis.
This procedure will produce an adequate but less precise fit of
the data. If samples have been diluted, the concentration read
from the standard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
The kit should not be used beyond the expiration date on the
kit label.
Do not mix or substitute reagents with those from other lots or
sources.
It is important that the Standard Diluent selected for the
standard curve be consistent with the samples being
assayed.
If samples generate values ​​higher than the highest standard,
dilute the samples with the appropriate Standard Diluent and
repeat the assay.
Any variation in Standard Diluent, operator, pipetting
technique, washing technique, incubation time or
temperature, and kit age can cause variation in binding.
This assay is designed to eliminate interference by soluble
receptors, binding proteins, and other factors present in
biological samples. Until all factors have been tested in the
Quantikine Immunoassay, the possibility of interference
cannot be excluded.
TECHNICAL HINTS
Centrifuge vials before opening to collect contents.
When mixing or reconstituting protein solutions, always avoid
foaming.
To avoid cross-contamination, change pipette tips between
additions of each standard level, between sample additions,
and between reagent additions. Also, use separate reservoirs
for each reagent.
When using an automated plate washer, adding a 30 second
soak period following the addition of wash buffer, and / or
rotating the plate 180 degrees between wash steps may
improve assay precision.
To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary.
Substrate Solution should remain colorless or light blue until
added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless or light
blue to gradations of blue.
Stop Solution should be added to the plate in the same order
as the Substrate Solution. The color developed in the wells
will turn from blue to yellow upon addition of the Stop Solution.
Wells that are green in color indicate that the Stop Solution
has not mixed thoroughly with the Substrate Solution.
Enzyme-linked immunoassay of rat hypersensitive heat shock protein 70 (HSP-70)
Kit instruction manual
This kit is for research use only
Detection range: 39 pg / ml-2500 pg / ml
Minimum detection limit: 9.75 pg / ml
Specificity: This kit can detect natural or recombinant rat HSP-70 at the same time, and is related to other
There is no cross-reactivity of the protein.
Validity: 6 months
Intended application: ELISA method for quantitative determination of rat serum, plasma or other related biological fluids
HSP-70 content.
Explanation
11. Preservation of the kit: unopened kits should be stored at 2-8 ° C; the microplate after opening should be dried
Store the agent together in an aluminum foil bag and store at 2-8 ° C. Only under this storage condition, the product has
It can be used normally within the validity period.
12. The concentrated washing liquid will have salt precipitation at low temperature, and it can be heated and dissolved in the water bath when diluted.
13. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
14. There may be some water-like substances in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not be wrong.
The test results have no effect.
Experimental principle
The microtiter plate is coated with purified antibody to make a solid-phase carrier, and then coated with anti-HSP-70 antibody
Add samples or standards, biotinylated anti-HSP-70 antibody, and HRP-labeled affinity to the wells in sequence
After thorough washing, it is developed with the substrate TMB. TMB is converted into peroxidase
Blue, and converted into the final yellow under the action of acid. The shade of color and HSP-70 in the sample
Was positively correlated. Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration.
Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Standard product (Standard): 2 bottles (lyophilized product).
3. Sample Diluent: 1 × 20ml / bottle.
4. Biotin-antibody Diluent: 1 × 10ml / bottle.
5. Horseradish peroxidase labeled avidin diluent (HRP-avidin Diluent) 1 × 10ml / bottle.
6. Biotin-antibody: 1 × 120μl / bottle (1: 100).
7. Horseradish peroxidase labeled avidin (HRP-avidin): 1 × 120μl / vial (1: 100).
8. Substrate solution (TMB Substrate): 1 × 10ml / bottle.
9. Wash Buffer 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop Solution: 1 × 10ml / bottle.
Reagents and equipment needed but not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Distilled water, volumetric flask, etc.
Collection and preservation of specimens
1. Serum: Whole blood samples should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000g for 20 minutes.
Take the supernatant for immediate detection; or aliquot and store the specimen at -20 ℃ or -80 ℃, but
Avoid repeated freezing and thawing. The thawed samples should be centrifuged again and then tested.
2. Plasma: EDTA or heparin can be used as anticoagulant, within 2-8 ° C within 30 minutes after specimen collection
Centrifuge at 1000 g for 15 minutes, take the supernatant for immediate detection; or aliquot and place the specimen in
Store at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing. The thawed sample should be centrifuged again, then
Detection.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000g for 20 minutes, immediately take the supernatant
Detection; or carry out sub-packaging and store the specimen at -20 ℃ or -80 ℃, but should avoid repeated freeze-thaw.
The thawed samples should be centrifuged again and then tested.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
Dilution principle of specimens:
First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor.
Only when it is diluted to the range of the standard curve, the test result is accurate. During the dilution process,
Keep detailed records. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N".
Standard dilution principle: 2 bottles, centrifuge at 6000-10000rpm for 30 seconds before use. each
Dilute the sample bottle to 1ml with the sample diluent before use, cover and let stand for more than 10 minutes, then invert repeatedly
/ Twist to help dissolve, its concentration is 2500 pg / ml, after serial dilution, dilute 2500 respectively
pg / ml, 1250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml, 78 pg / ml, 39 pg / ml, sample
The product dilution is directly used as the standard concentration of 0 pg / ml, prepared within 15 minutes before use, discarded after use, and
Use freshly configured standards for the second test.
If preparing 1250 pg / ml standard: take 0.5ml (not less than 0.5ml) 2500 pg / ml of the above
The standard is added to the Eppendorf tube containing 0.5ml of sample diluent and mixed well.
And so on.
Dilution principle of biotinylated antibody:
Before opening the cap, centrifuge to collect the solution on the bottle wall. Diluted with biotin-labeled antibody dilution before use
Before the dilution, according to the pre-calculated total amount required for each experiment (100μl per well), the actual
When preparing, 0.1-0.2ml should be prepared. For example, 10μl biotin-labeled antibody plus 990μl biotin-labeled antibody
Prepare the ratio of diluent, mix gently, and prepare within one hour before use.
Dilution principle of horseradish peroxidase-labeled avidin:
Before opening the cap, centrifuge to collect the solution on the bottle wall. Labeled affinity with horseradish peroxidase before use
Diluent dilution, according to the pre-calculated total amount required for each experiment before dilution (per well
100μl), more 0.1-0.2ml should be prepared in actual preparation. Such as 10μl horseradish peroxidase labeled avidin
Add 990μl of horseradish peroxidase-labeled avidin dilution, mix gently, and use
Prepared within the first hour.
Steps
Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they must be mixed well.
Try to avoid blistering. A standard curve should be made for each test. If the sample concentration is too high, use the sample
The dilution solution is diluted so that the sample conforms to the detection range of the kit. When loading samples, the gun head should be directly
On the quasi-liquid surface, do not apply sample along the hole wall.
4. Serum and plasma samples are diluted 1:20 times with sample diluent for testing, the specific operations are as follows
Bottom: Add 15μl of sample to 285μl of sample dilution (1:20 dilution) and mix well. Get
The result is the 1: 20-fold diluted sample.
5. Add sample: set blank hole, standard hole and sample hole to be tested. Add 100μl of sample diluent to blank wells,
Add 100μl of the standard product or the sample to be tested to the remaining hole, be careful not to have bubbles, add the sample to the sample
At the bottom of the well of the microtiter plate, try not to touch the wall of the well, gently shake and mix well.
Incubate at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
6. Discard the liquid and spin dry without washing. Add 100μl of biotinylated antibody working solution to each well (take 1μl
Prepare a ratio of biotin-labeled antibody plus 99μl of biotin-labeled antibody dilution, mix gently,
Prepared within one hour before use), 37 ℃, 60 minutes.
7. After 60 minutes of incubation, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
8. Add horseradish peroxidase-labeled avidin working solution to each well (same as biotin-labeled antibody working solution)
100 μl, 37 ° C, 60 minutes.
9. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
10. Add 90 μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, the standard is visible to the naked eye)
The first 3-4 holes of the quasi product have a clear gradient blue, and the latter 3-4 holes are not obvious, and can be terminated).
11. Add 50μl of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). Stop solution
The order of addition should be the same as the order of addition of substrate solution. In order to ensure the accuracy of the experimental results, the bottom
The stop solution should be added as soon as possible after the reaction time.
12. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Stop solution
Test within 15 minutes.
Experimental remarks
1. When using the kit for the first time, the user should centrifuge various reagent tubes for a few minutes
Tube bottom.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate is added at the end to dissolve
Solution and stop solution. Use this hole to adjust the OD value to zero first during measurement.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.
Cover or cover.
4. Store unused microplates or reagents at 2-8 ° C. Standards, biotinylated antibodies
Working solution, horseradish peroxidase-labeled avidin working solution, please use according to the required amount. please
Do not reuse diluted standards, biotinylated antibody working solution, or horseradish peroxide
Enzyme-labeled avidin working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
Washing method
Manual plate washing method: aspirate (do not touch the wall) or shake off the liquid in the enzyme plate; on the experimental table
Paved with several layers of absorbent paper, and slamming the microtiter plate down several times; put the recommended wash buffer at least 0.3ml
Fill the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Calculation
Please download the professional software "Curve Exert 1.3" from our website and follow the prompts to make a standard curve.
Taking the concentration of the standard as the abscissa (logarithmic coordinate), the OD value is the ordinate (common coordinate),
Draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample;
Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression of the standard curve
Formula, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor, that is
The actual concentration of the sample.
Precautions
1. These operating instructions apply to 48T kits, enzyme-linked plates, standards, and biotin in the 48T kits
Labeled antibody and horseradish peroxidase labeled avidin halved.
2. When mixing the protein solution, it should be as gentle as possible to avoid foaming.
3. The washing process is very important. Insufficient washing can easily cause false positives.
4. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
5. Please make a standard curve at the same time every measurement.
6. If the content of the substance to be tested in the specimen is too high, please dilute it before measuring, please multiply it by the dilution when calculating
multiple.
7. When preparing standard products and testing solution working fluids, please prepare with corresponding diluent, not to be confused.
8. Please keep the substrate away from light.
9. Do not replace the reagents in the kit with reagents from other manufacturers.
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