96 Wells Customized Mouse Elisa Kit Vascular Endothelial Cell Growth Factor
Brand Name:BT Lab
Certification:CE, ISO9001:2005, MSDS
Model Number:Cat.No E0114Mo
Minimum Order Quantity:Negotiation
Delivery Time:1-3 business days, bulk order within one week
Payment Terms:Western Union, T/T
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Product Details
96 Wells Customized Mouse Vascular Endothelial Cell Growth Factor
ELISA Kit
Cat.No E0114Mo
Standard Curve Range: 5ng/L - 1800ng/L
Sensitivity: 2.48ng/L
Size: 96 wells
Intended Use
This sandwich kit is for the accurate quantitative detection of
Mouse Vascular Endothelial Cell Growth Factor (also known as VEGF)
in serum, plasma, cell culture supernates, cell lysates, tissue
homogenates.
Assay Principle
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate
has been pre-coated with Mouse VEGF antibody. VEGF present in the
sample is added and binds to antibodies coated on the wells. And
then biotinylated Mouse VEGF Antibody is added and binds to VEGF in
the sample. Then Streptavidin-HRP is added and binds to the
Biotinylated VEGF antibody. After incubation unbound
Streptavidin-HRP is washed away during a washing step. Substrate
solution is then added and color develops in proportion to the
amount of Mouse VEGF. The reaction is terminated by addition of
acidic stop solution and absorbance is measured at 450 nm.
Precautions
- Prior to use, the kit and sample should be warmed naturally to room temperature 30 minutes.
- This instruction must be strictly followed in the experiment.
- Once the desired number of strips has been removed, immediately reseal the bag to protect the remain from deterioration. Cover all reagents when not in use.
- Make sure pipetting order and rate of addition from well-to-well when pipetting reagents.
- Pipette tips and plate sealer in hand should be clean and disposable to avoid cross-contamination.
- Avoid using the reagents from different batches together.
- Substrate solution B is sensitive to light, don’t expose substrate solution B to light for a long time.
- Stop solution contains acid. Please wear eye, hand and skin protection when using this material. Avoid contact of skin or mucous membranes with kit reagent.
- The kit should not be used beyond the expiration date.
Specimen Collection
Serum Allow serum to clot for 10-20 minutes at room temperature.
Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant.
Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C
within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for
approximately 20 minutes. When collecting pleuroperitoneal fluid
and cerebrospinal fluid, please follow the procedures
above-mentioned.
Cell Culture Supernatant Collect by sterile tubes when examining secrete components.
Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect
the supernatants carefully. When examining the components within
the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the
cell concentration of approximately 1 million/ml. Damage cells
through repeated freeze-thaw cycles to let out the inside
components. Centrifuge at 2000-3000 RPM for approximately 20
minutes.
Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly
and weigh before homogenization. Mince tissues and homogenize them
in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or
freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20
minutes.
*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample
matrix interference may falsely depress the specificity and
accuracy of the assay.
Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (2000ng/L) with 120μl of
standard diluent to generate a 1000ng/L standard stock solution.
Allow the standard to sit for 15 mins with gentle agitation prior
to making dilutions. Prepare duplicate standard points by serially
diluting the standard stock solution (1000ng/L) 1:2 with standard
diluent to produce 500ng/L, 250ng/L, 125ng/L and 62.5ng/L
solutions. Standard diluent serves as the zero standard(0 ng/L).
Any remaining solution should be frozen at -20°C and used within
one month. Dilution of standard solutions suggested are as follows:
| 1000ng/L | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
| 500ng/L | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
| 250ng/L | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
| 125ng/L | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
| 62.5ng/L | Standard No.1 | 120μl Standard No.2 + 120μl Standard Diluent |
| Standard Concentration | Standard No.5 | Standard No.4 | Standard No.3 | Standard No.2 | Standard No.1 |
| 2000ng/L | 1000ng/L | 500ng/L | 250ng/L | 125ng/L | 62.5ng/L |
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or
distilled water to yield 500 ml of 1x Wash Buffer. If crystals have
formed in the concentrate, mix gently until the crystals have
completely dissolved.
Summary
1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour
at 37°C.
3. Wash the plate 5 times.
4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.
5. Add stop solution and color develops.
6. Read the OD value within 10 minutes.
Calculation of Result
Construct a standard curve by plotting the average OD 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. These calculations can be best performed with
computer-based curve-fitting software and the best fit line can be
determined by regression analysis.
96 Wells Customized Mouse Elisa Kit Vascular Endothelial Cell Growth Factor
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