大鼠磷酸化蛋白激酶B(P-PKB)ELISA試劑盒使用說明

1
Rat Phosphorylated Protein Kinase B (p-PKB)
ELISA Kit

For the quantitative determination of rat phosphorylated protein kinase B
(p-PKB) concentrations in serum, plasma, tissue homogenates.
This package insert must be read in its entirety before using this product.

In order to obtain higher efficiency service, please ready to supply the lot number 
of the kit to us (found on the outside of the box).2
PRINCIPLE OF THE ASSAY
This assay employs the quantitative sandwich enzyme immunoassay technique. 
Antibody specific for p-PKB has been pre-coated onto a microplate. Standards 
and samples are pipetted into the wells and any p-PKB present is bound by the 
immobilized antibody. After  removing  any unbound substances, a 
biotin-conjugated antibody specific for  p-PKB is added to the wells. After
washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells.
Following a wash to remove any unbound avidin-enzyme reagent, a substrate 
solution is added to the wells and color develops in proportion to the amount of 
p-PKB bound in the initial step. The color development is stopped and the 
intensity of the color is measured.
DETECTION RANGE
3.12 pg/ml-200 pg/ml.
SENSITIVITY
The minimum detectable dose of rat p-PKB is typically less than 0.78 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. It was 
determined the mean O.D value of 20 replicates of the zero standard added by 
their three standard deviations.
SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of rat p-PKB. 
No significant cross-reactivity or interference between rat p-PKB and analogues 
was observed.
Note: Limited by current skills and knowledge, it is impossible for us to complete 
the cross-reactivity detection between rat p-PKB and all the analogues, therefore,
cross reaction may still exist.3
PRECISION
Intra-assay Precision (Precision within an assay): CV%<8%
Three samples of known concentration were tested twenty times on one plate to 
assess.
Inter-assay Precision (Precision between assays): CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
LIMITATIONS OF THE PROCEDURE
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC 
PROCEDURES.
? 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.
? If samples generate values higher than the highest standard, dilute the 
samples with Sample Diluent and repeat the assay.
? Any variation in  Sample 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 Immunoassay, the possibility of 
interference cannot be excluded.4
MATERIALS PROVIDED
Reagents Quantity
Assay plate (12 x 8 coated Microwells) 1(96 wells)
Standard (Freeze dried) 2
Biotin-antibody (100 x concentrate) 1 x 120 μl
HRP-avidin (100 x concentrate) 1 x 120 μl
Biotin-antibody Diluent   1 x 10 ml
HRP-avidin Diluent    1 x 10 ml
Sample Diluent   2 x 20 ml
Wash Buffer (25 x concentrate) 1 x 20 ml
TMB Substrate  1 x 10 ml
Stop Solution   1 x 10 ml
Adhesive Strip (For 96 wells) 4
Instruction manual 1
STORAGE
Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date 
Opened kit
Coated assay 
plate
May be stored for up to 1 month at 2 - 8°C. 
Try to keep it in a sealed aluminum foil bag,
and avoid the damp.
Standard May be stored for up to 1 month at 2 - 8° C. If 
don’t make recent use, better keep it store at 
-20°C.
Biotin-antibody 
HRP-avidin
Biotin-antibody 
Diluent
May be stored for up to 1 month at 2 - 8°C.
HRP-avidin 
Diluent
Sample 
Diluent
Wash Buffer
TMB 
Substrate
Stop Solution
*Provided this is within the expiration date of the kit.5
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, with the 
correction wavelength set at 540 nm or 570 nm.
? An incubator which can provide stable incubation conditions up to 
37°C±0.5°C.
? Squirt bottle, manifold dispenser, or automated microplate washer.
? Absorbent paper for blotting the microtiter plate.
? 100ml and 500ml graduated cylinders.
? Deionized or distilled water.
? Pipettes and pipette tips.
? Test tubes for dilution.
PRECAUTIONS
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, 
and clothing protection when using this material.6
SAMPLE COLLECTION AND STORAGE
? Serum Use a serum separator tube (SST) and allow samples to clot for 
two hours at room temperature or overnight at 4°C before centrifugation 
for  15 minutes at 1000  ×g. Remove serum and assay immediay or 
aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw 
cycles.
? Plasma Collect plasma using EDTA, or heparin as an anticoagulant. 
Centrifuge for 15 minutes at 1000  ×g at 2-8°C within 30 minutes of 
collection. Assay immediay or aliquot and store samples at  -20°C or
-80°C. Avoid repeated freeze-thaw cycles.
? Tissue Homogenates 100mg tissue was rinsed with 1XPBS, 
homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two 
freeze-thaw cycles were performed to break the cell membranes, the 
homogenates were centrifuged for 5 minutes at 5000 x g, 2  - 8°C. The 
supernate was assayed and removed immediay. Alternatively, aliquot 
and store samples at -20°C or -80°C. Centrifuge the sample again after 
thawing before the assay. Avoid repeated freeze-thaw cycles.7
SAMPLE PREPARTION
Recommend to dilute the serum or plasma samples with Sample Diluent (1:200) 
before test. The suggested 200-fold dilution can be achieved by adding 5μl 
sample to 45μl of Sample Diluent. Complete the 200-fold dilution by adding15μl 
of this solution to 285μl of Sample Diluent. The recommended dilution factor is 
for reference only. The optimal dilution factor should be determined by users 
according to their particular experiments.
Note:
1. CUSABIO 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 
samples in advance.
2. Samples to be used within 5 days may be stored at 2-8°C, otherwise 
samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid 
loss of bioactivity and contamination.
3. Grossly hemolyzed samples are not suitable for use in this assay.
4. If the samples are not indicated in the manual, a preliminary experiment to 
determine the validity of the kit is necessary. 
5. 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.
6. Tissue or cell extraction samples prepared by chemical lysis buffer may 
cause unexpected ELISA results due to the impacts of certain chemicals.
7. 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.
8. 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.
9. Fresh samples without long time storage are recommended for the test. 
Otherwise, protein degradation and denaturalization may occur in those 
samples and finally lead to wrong results.8
REAGENT PREPARATION
Note: 
? Kindly use graduated containers to prepare the reagent. Please don't 
prepare the reagent directly in the Diluent vials provided in the kit.
? Bring all reagents to room temperature (18-25°C) before use for 30min.
? Prepare fresh standard for each assay. Use within 4 hours and discard 
after use.
? Making serial dilution in the wells directly is not permitted. 
? Please carefully reconstitute Standards according to the instruction, and 
avoid foaming and mix gently until the crystals have compley dissolved. 
To minimize imprecision caused by pipetting, use small volumes and 
ensure that pipettors are calibrated. It is recommended to suck more than 
10μl for once pipetting.
? Distilled water is recommended to be used to make the preparation for 
reagents or samples. Contaminated water or  container for reagent 
preparation will influence the detection result.
1. Biotin-antibody (1x) - Centrifuge the vial before opening. 
Biotin-antibody requires a 100-fold dilution. A suggested 100-fold dilution 
is 10 μl of Biotin-antibody + 990 μl of Biotin-antibody Diluent.
2. HRP-avidin (1x) - Centrifuge the vial before opening.
HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 
μl of HRP-avidin + 990 μl of HRP-avidin Diluent.
3. Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to    
room temperature and mix gently until the crystals have compley 
dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or 
distilled water to prepare 500 ml of Wash Buffer (1 x).9
4. Standard 
Centrifuge the standard vial at 6000-10000rpm for 30s. 
Reconstitute the  Standard with 1.0 ml of  Sample Diluent. Do not 
substitute other diluents. This reconstitution produces a stock solution of 
200 pg/ml. Mix the standard to ensure complete reconstitution and allow 
the standard to sit for a minimum of 15 minutes with gentle agitation prior 
to making dilutions. 
Pipette 250 μl of Sample Diluent into each tube (S0-S6). Use the stock 
solution to produce a 2-fold dilution series (below). Mix each tube 
thoroughly before the next transfer. The undiluted Standard serves as the 
high standard (200 pg/ml). Sample Diluent serves as the zero standard (0 
pg/ml).
Tube S7 S6 S5 S4 S3 S2 S1 S0
pg/ml 200 100 50 25 12.5 6.25 3.12 010
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. Centrifuge 
the sample again after thawing before the assay. It is recommended that all 
samples and standards be assayed in duplicate. 
1. Prepare all reagents, working standards, and samples as directed in the 
previous sections.
2. Refer to the Assay Layout Sheet to determine the number of wells to be 
used and put any remaining wells and the desiccant back into the pouch 
and seal the ziploc, store unused wells at 4°C.
3. Add 100μl of standard and sample per well. Cover with the adhesive strip
provided. Incubate for 2 hours at 37°C. A plate layout is provided to record 
standards and samples assayed.
4. Remove the liquid of each well, don’t wash. 
5. Add 100μl of  Biotin-antibody (1x)  to each well. Cover with a new 
adhesive strip. Incubate for 1 hour at 37°C. (Biotin-antibody (1x) may 
appear cloudy. Warm up to room temperature and mix gently until solution 
appears uniform.)
6. Aspirate each well and wash, repeating the process two times for a total of 
three washes. Wash by filling each well with Wash Buffer (200μl) using a 
squirt bottle, multi-channel pipette, manifold dispenser, or autowasher,
and let it stand for 2 minutes, complete removal of liquid at each step is 
essential to good performance. After the last wash, remove any remaining 
wash Buffer by aspirating ordecanting. Invert the plate and blot it against 
clean paper towels.
7. Add 100μl of HRP-avidin (1x) to each well. Cover the microtiter plate with 
a new adhesive strip. Incubate for 1 hour at 37°C.
8. Repeat the aspiration/wash process for five times as in step 6.
9. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at 
37°C. Protect from light.
10. Add 50μl of Stop Solution to each well,  gently tap the plate to ensure 
thorough mixing.11
11. Determine the optical density of each well within  5 minutes, using a 
microplate reader set to 450 nm. If wavelength correction is available, set 
to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the 
readings at 450 nm. This subtraction will correct for optical imperfections 
in the plate. Readings made directly at 450 nm without correction may be 
higher and less accurate.
*Samples may require dilution. Please refer to Sample Preparation section.
Note:
1. The final experimental results will be closely related to validity of the 
products, operation skills of the end users and the experimental 
environments. 
2. Samples or reagents addition: Please use the freshly prepared Standard. 
Please carefully add samples to wells and mix gently to avoid foaming. Do 
not touch the well wall as possible. For each step in the procedure, total 
dispensing time for addition of reagents or samples to the assay plate 
should not exceed 10 minutes. This will ensure equal elapsed time for 
each pipetting step, without interruption. Duplication of all standards and 
specimens, although not required, is recommended. 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.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers 
during incubation steps is necessary. Do not allow wells to sit uncovered 
for extended periods between incubation steps. Once reagents have been 
added to the well strips, DO NOT let the strips DRY at any time during the 
assay. Incubation time and temperature must be observed.
4. Washing: The wash procedure is critical. Complete removal of liquid at 
each step is essential to good performance. After the last wash, remove 
any remaining Wash Solution by aspirating or decanting and remove any 
drop of water and fingerprint on the bottom of the plate. Insufficient 
washing will result in poor precision and falsely elevated absorbance 
reading. 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.12
5. Controlling of reaction time: Observe the change of color after adding TMB 
Substrate (e.g. observation once every 10 minutes), TMB Substrate
should change from colorless or light blue to gradations of blue. If the color 
is too deep, add Stop Solution in advance to avoid excessively strong 
reaction which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated.  TMB Substrate should remain 
colorless or light blue until added to the plate. Please protect it from light.
7. Stop Solution should be added to the plate in the same order as the TMB 
Substrate. 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 TMB Substrate.13
ASSAY PROCEDURE SUMMARY
*Samples may require dilution. Please refer to Sample Preparation section.14
CALCULATION OF RESULTS
Using the professional soft "Curve Expert 1.3" to make a standard curve is 
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard 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 x-axis against the concentration on the y-axis and draw a best fit curve 
through the points on the graph. The data may be linearized by plotting the log of 
the p-PKB concentrations versus the log of the O.D. 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.

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