Human SPAG9/C-Jun-amino-terminal kinase-interacting protein 4 ELISA Kit (HUFI01123)

Product Name:	Human SPAG9/C-Jun-amino-terminal kinase-interacting protein 4 ELISA Kit
Product Code:	HUFI01123
Size:	96 Assays
Alias:	SPAG9, JIP-4, JNK-interacting protein 4, C-Jun-amino-terminal kinase-interacting protein 4, Protein highly expressed in testis, PHET, ProlifeRation-inducing protein 6, Mitogen-activated protein kinase 8-interacting protein 4, JNK-associated leucine-zipper protein, JLP, Sperm-specific protein, Sperm-associated antigen 9, Human lung cancer oncogene 6 protein, HLC-6
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human SPAG9 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human SPAG9 and the recovery rates were calculated by comparing the measured value to the expected amount of Human SPAG9 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-104	96
EDTA plasma(n=5)	85-104	93
UFH plasma(n=5)	91-104	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human SPAG9 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)	89-93%	87-104%	93-102%
EDTA plasma(n=5)	88-100%	82-96%	83-97%
UFH plasma(n=5)	83-99%	84-97%	80-97%

CV(%):

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

Component	Quantity	Storage
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	-

Other materials and equipment required:

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

O60271

UniProt Protein Function:	JIP4: The JNK-interacting protein (JIP) group of scaffold proteins selectively mediates JNK signaling by aggregating specific components of the MAPK cascade to form a functional JNK signaling module. Isoform 5 may play a role in spermatozoa-egg- interaction. Homooligomer. Interacts with MAX, MAPK8, MAPK9, MAPK10, MAPK14, MAP3K3, MYC, KNS2 and MAP2K4. Interaction with KNS2 is important in the formation of ternary complex with MAPK8. Interacts with NFKB1. Isoform 3 is increased in systemic sclerosis fibroblasts. Isoform 5 is expressed only in testis on the round spermatids of stage I, II and II. Isoform 5 is absent in spermatogonia and spermatocyte. Isoform 3 is expressed in testis. Isoform 4 is expressed in testis and in acute myeloid leukemia (AML) patients. Belongs to the JIP scaffold family. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Adaptor/scaffold; Cancer Testis Antigen (CTA) Chromosomal Location of Human Ortholog: 17q21.33 Cellular Component: cytoplasm; cytosol; integral to membrane; microtubule organizing center Molecular Function:JUN kinase binding; kinesin binding; MAP-kinase scaffold activity; protein binding; receptor signaling complex scaffold activity Biological Process: activation of JNK activity; positive regulation of cell migration; positive regulation of muscle cell differentiation; retrograde transport, endosome to Golgi; spermatogenesis
NCBI Summary:	This gene encodes a member of the cancer testis antigen gene family. The encoded protein functions as a scaffold protein that structurally organizes mitogen-activated protein kinases and mediates c-Jun-terminal kinase signaling. This protein also binds to kinesin-1 and may be involved in microtubule-based membrane transport. This protein may play a role in tumor growth and development. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Oct 2011]
UniProt Code:	O60271
NCBI GenInfo Identifier:	94717648
NCBI Gene ID:	9043
NCBI Accession:	O60271.4
UniProt Secondary Accession:	O60271,O60905, Q3KQU8, Q3MKM7, Q86WC7, Q86WC8, Q8IZX7 Q96II0, Q9H811, A6H8U5, A8MSX0, B4DHH2,
UniProt Related Accession:	O60271
Molecular Weight:	128,607 Da
NCBI Full Name:	C-Jun-amino-terminal kinase-interacting protein 4
NCBI Synonym Full Names:	sperm associated antigen 9
NCBI Official Symbol:	SPAG9
NCBI Official Synonym Symbols:	JLP; CT89; HLC4; HLC6; JIP4; PHET; PIG6; HLC-6; JIP-4
NCBI Protein Information:	C-Jun-amino-terminal kinase-interacting protein 4
UniProt Protein Name:	C-Jun-amino-terminal kinase-interacting protein 4
UniProt Synonym Protein Names:	Cancer/testis antigen 89; CT89; Human lung cancer oncogene 6 protein; HLC-6; JNK-associated leucine-zipper protein; JLP; Mitogen-activated protein kinase 8-interacting protein 4; Proliferation-inducing protein 6; Protein highly expressed in testis; PHET; Sperm surface protein; Sperm-associated antigen 9; Sperm-specific protein; Sunday driver 1
Protein Family:	C-Jun-amino-terminal kinase-interacting protein
UniProt Gene Name:	SPAG9
UniProt Entry Name:	JIP4_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 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.

Step	Protocol
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 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.
Plasma	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.
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 & 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.
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.