Rat IL-2R alpha/CD25 ELISA Kit (RTFI00908)

Product Name:	Rat IL-2R alpha/CD25 ELISA Kit
Product Code:	RTFI00908
Size:	96 Assays
Target:	Rat IL-2Ralpha
Alias:	IL-2Ralpha, CD25, Interleukin 2 Receptor Alpha
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat IL-2Ralpha and the recovery rates were calculated by comparing the measured value to the expected amount of Rat IL-2Ralpha in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-105	92
EDTA plasma(n=5)	85-103	93
UFH plasma(n=5)	86-104	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat IL-2Ralpha and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-100%	85-103%	85-103%
EDTA plasma(n=5)	84-101%	84-100%	83-96%
UFH plasma(n=5)	81-100%	81-97%	81-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P26897

UniProt Protein Function:	IL2RA: Receptor for interleukin-2. Non-covalent dimer of an alpha and a beta subunit. IL2R exists in 3 different forms: a high affinity dimer, an intermediate affinity monomer (beta subunit), and a low affinity monomer (alpha subunit). The high and intermediate affinity forms also associate with a gamma subunit.
UniProt Protein Details:	Protein type:Membrane protein, integral Chromosomal Location of Human Ortholog: 10p15-p14 Cellular Component: integral to membrane; plasma membrane; external side of plasma membrane Molecular Function:interleukin-2 receptor activity; drug binding; interleukin-2 binding Biological Process: Notch signaling pathway; regulation of T cell homeostatic proliferation; apoptosis; negative regulation of immune response; negative regulation of defense response to virus; positive regulation of activated T cell proliferation; cell proliferation; negative regulation of T cell proliferation; inflammatory response to antigenic stimulus; cell surface receptor linked signal transduction; positive regulation of T cell differentiation; negative regulation of inflammatory response; immune response; inflammatory response; activated T cell apoptosis Disease: Interleukin 2 Receptor, Alpha, Deficiency Of; Diabetes Mellitus, Insulin-dependent, 10
NCBI Summary:	The interleukin 2 (IL2) receptor alpha (IL2RA) and beta (IL2RB) chains, together with the common gamma chain (IL2RG), constitute the high-affinity IL2 receptor. Homodimeric alpha chains (IL2RA) result in low-affinity receptor, while homodimeric beta (IL2RB) chains produce a medium-affinity receptor. Normally an integral-membrane protein, soluble IL2RA has been isolated and determined to result from extracellular proteolyisis. Alternately-spliced IL2RA mRNAs have been isolated, but the significance of each is presently unknown. Mutations in this gene are associated with interleukin 2 receptor alpha deficiency.[provided by RefSeq, Nov 2009]
UniProt Code:	P26897
NCBI GenInfo Identifier:	4557667
NCBI Gene ID:	3559
NCBI Accession:	NP_000408.1
UniProt Secondary Accession:	P26897,P26897,
UniProt Related Accession:	P01589
Molecular Weight:	30,819 Da
NCBI Full Name:	interleukin-2 receptor subunit alpha
NCBI Synonym Full Names:	interleukin 2 receptor, alpha
NCBI Official Symbol:	IL2RA
NCBI Official Synonym Symbols:	CD25; IL2R; TCGFR; IDDM10
NCBI Protein Information:	interleukin-2 receptor subunit alpha; p55; IL2-RA; IL-2-RA; TAC antigen; IL-2R subunit alpha; IL-2 receptor subunit alpha
UniProt Protein Name:	Interleukin-2 receptor subunit alpha
UniProt Synonym Protein Names:	TAC antigen; p55
UniProt Gene Name:	IL2RA
UniProt Entry Name:	IL2RA_HUMAN

Step	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 10-20 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.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.