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Rat Smad7 / MADH7 ELISA Kit

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SKU:
RTFI01135
$667

Description

Rat Smad7 / MADH7 ELISA Kit - Information

The ELISA Genie Rat Smad7 / MADH7 ELISA Kit can assay for Rat Smad7 / MADH7 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Rat Smad7 / MADH7 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Rat Smad7 / MADH7 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Rat Smad7 / MADH7 ELISA Kit - Data

Description

Antagonist of signaling by TGF-beta (transforming growth factor) type 1 receptor superfamily members; has been shown to inhibit TGF-beta (Transforming growth factor) and activin signaling by associating with their receptors thus preventing SMAD2 access. Functions as an adapter to recruit SMURF2 to the TGF-beta receptor complex. Also acts by recruiting the PPP1R15A-PP1 complex to TGFBR1, which promotes its dephosphorylation. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.

Post-Translational Modification

Phosphorylation on Ser-249 does not affect its stability, nuclear localization or inhibitory function in TGFB signaling; however it affects its ability to regulate transcription. Phosphorylated by PDPK1. Ubiquitinated by WWP1. Polyubiquitinated by RNF111, which is enhanced by AXIN1 and promotes proteasomal degradation. In response to TGF-beta, ubiquitinated by SMURF1; which promotes its degradation. Acetylation prevents ubiquitination and degradation mediated by SMURF1.

Uniprot ID O88406
Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Smad7 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.313-20ng/ml

Sensitivity

< 0.188ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Smad7 and the recovery rates were calculated by comparing the measured value to the expected amount of Smad7 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 89-100 96
EDTA plasma(n=5) 87-102 94
UFH plasma(n=5) 86-104 96
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Smad7 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 86-102% 90-105% 89-105% 85-104%
EDTA plasma(n=5) 82-100% 91-101% 84-97% 82-96%
UFH plasma(n=5) 80-88% 81-98% 83-100% 80-96%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Datasheets

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Protocol

Sandwich ELISA Protocol

The below protocol is a sample protocol for a sandwich ELISA using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of analyte present in their sample. This can be very useful when looking for increases in protein concentration, phosphorylation of proteins or decreases on protein concentrations.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at room temperature (37 °C). When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

ELISA Kit Technical Manual

Procedure:

1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 15-30 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.
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Additional Information

Reactivity:
Rat
ELISA Type:
Sandwich
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