Rat HSP40 ELISA Kit - Information
The ELISA Genie Rat HSP40 ELISA Kit can assay for Rat HSP40 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Rat HSP40 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 HSP40 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 HSP40 ELISA Kit - Data
|Detection method || |
Sandwich ELISA Double Antibody
|Application || |
This immunoassay kit allows for the in vitro quantitative determination of HSP-40 concentrations in serum plasma and other biological fluids.
|Size || |
|Range || |
|Sensitivity || |
|Storage || |
4'C for 6 months
|Recovery || |
Matrices listed below were spiked with certain level of HSP-40 and the recovery rates were calculated by comparing the measured value to the expected amount of HSP-40 in samples.
|Matrix ||Recovery range(%) ||Average(%) |
|serum(n=5) ||85-95 ||90 |
|EDTA plasma(n=5) ||85-100 ||91 |
|UFH plasma(n=5) ||90-103 ||97 |
|Linearity || |
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of HSP-40 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) ||85-100% ||85-98% ||88-104% ||86-104% |
|EDTA plasma(n=5) ||85-101% ||82-100% ||83-99% ||82-99% |
|UFH plasma(n=5) ||80-97% ||80-100% ||81-94% ||82-100% |
|CV(%) || |
|Note || |
For Research Use Only
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.
|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.|