ねずみ IL-1A/IL-1 alpha ELISA Kit

商品名:	Mouse IL-1A / IL-1 alpha ELISA Kit
製品コード:	MOFI00057
サイズ:	96 Assays
エイリアス:	IL-1alpha, IL-1alpha, IL1A, IL1-A, IL1, IL1F1, Preinterleukin 1 Alpha, Hematopoietin-1, Pro-Interleukin-1-Alpha
検出方法:	Sandwich ELISA
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Mouse IL-1alpha concentrations in serum plasma and other biological fluids.
感度:	9.375pg/ml
範囲:	15.625-1000pg/ml
保管所:	4°C for 6 months
ノート:	For Research Use Only

回復:

Matrices listed below were spiked with certain level of Mouse IL-1alpha and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse IL-1alpha in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	95-105	98
EDTA plasma(n=5)	93-104	98
UFH plasma(n=5)	86-100	95

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse IL-1alpha 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)	88-102%	88-101%	92-104%
EDTA plasma(n=5)	82-98%	84-90%	87-99%
UFH plasma(n=5)	81-100%	86-95%	80-100%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

成分	量	保管所
ELISA Microplate (Dismountable)	8-12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

必要なその他の材料と設備：

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P01582

UniProt Protein Function:	IL1A: Produced by activated macrophages, IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. IL-1 proteins are involved in the inflammatory response, being identified as endogenous pyrogens, and are reported to stimulate the release of prostaglandin and collagenase from synovial cells. Belongs to the IL-1 family.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Cytokine Cellular Component: extracellular space; cell surface; plasma membrane; extracellular region; cytosol Molecular Function:interleukin-1 receptor binding; copper ion binding; cytokine activity Biological Process: positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of mitosis; positive regulation of interleukin-2 biosynthetic process; cytokine and chemokine mediated signaling pathway; germ cell programmed cell death; positive regulation of JNK cascade; positive regulation of interleukin-6 production; fever; positive regulation of stress-activated MAPK cascade; negative regulation of cell proliferation; positive regulation of angiogenesis; connective tissue replacement during inflammatory response; response to copper ion; positive regulation of cell division; keratinization; positive regulation of prostaglandin secretion; immune response; positive regulation of transcription from RNA polymerase II promoter; regulation of sensory perception of pain; inflammatory response; positive regulation of cytokine secretion
UniProt Code:	P01582
NCBI GenInfo Identifier:	124298
NCBI Gene ID:	16175
NCBI Accession:	P01582.1
UniProt Related Accession:	P01582
Molecular Weight:	31,023 Da
NCBI Full Name:	Interleukin-1 alpha
NCBI Synonym Full Names:	interleukin 1 alpha
NCBI Official Symbol:	Il1a
NCBI Official Synonym Symbols:	Il-1a
NCBI Protein Information:	interleukin-1 alpha; IL-1 alpha
UniProt Protein Name:	Interleukin-1 alpha
Protein Family:	Interleukin
UniProt Gene Name:	Il1a
UniProt Entry Name:	IL1A_MOUSE

*ノート:プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。

ステップ	手順
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.

ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。

サンプルタイプ	プロトコル
血清:	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 clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
プラズマ:	Collect plasma using EDTA or heparin as an anticoagulant. 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.
尿および脳脊髄液:	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.
細胞培養上清:	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.
細胞溶解物:	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.
組織ホモジネート:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 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.
組織溶解物:	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.
母乳:	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.