Human Sorting nexin-9 (SNX9) ELISA Kit (HUEB1153)

Product Name:	Human Sorting nexin-9 (SNX9) ELISA Kit
SKU:	HUEB1153
Size:	96T
Target:	Human Sorting nexin-9 (SNX9)
Synonyms:	SH3 and PX domain-containing protein 1, SH3 and PX domain-containing protein 3A, Protein SDP1, SH3PX1, SH3PXD3A
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.12ng/ml

Intra CV:

5.2%

Inter CV:

9.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	86-98%	98-108%	100-110%	109-121%
EDTA Plasma(N=5)	91-101%	105-115%	93-103%	115-125%
Heparin Plasma(N=5)	108-117%	101-109%	105-117%	88-99%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	100	94-106
Plasma	102	96-108

Function:

Involved in endocytosis and intracellular vesicle trafficking, both during interphase and at the end of mitosis. Required for efficient progress through mitosis and cytokinesis. Required for normal formation of the cleavage furrow at the end of mitosis. Plays a role in endocytosis via clathrin-coated pits, but also clathrin-independent, actin-dependent fluid-phase endocytosis. Plays a role in macropinocytosis. Promotes internalization of TNFR. Promotes degradation of EGFR after EGF signaling. Stimulates the GTPase activity of DNM1. Promotes DNM1 oligomerization. Promotes activation of the Arp2/3 complex by WASL, and thereby plays a role in the reorganization of the F-actin cytoskeleton. Binds to membranes enriched in phosphatidylinositol 4,5-bisphosphate and promotes membrane tubulation. Has lower affinity for membranes enriched in phosphatidylinositol 3-phosphate.

Uniprot:	Q9Y5X1
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Sorting nexin-9
Sub Unit:	Homodimer, and homooligomer. Heterodimer with SNX18. Interacts with ITCH. Interacts (via SH3 domain) with TNK2, WASL and ARP3. Identified in a complex with TNK2 and clathrin heavy chains. Identified in a complex with the AP-2 complex, clathrin and DNM2. Interacts (via SH3 domain) with DNM1 and DNM2. Identified in an oligomeric complex containing DNM1 and SNX9. Interacts with ADAM9 and ADAM15 cytoplasmic tails.
Subcellular Location:	Cytoplasmic vesicle membrane Peripheral membrane protein Cytoplasmic side Cell membrane Peripheral membrane protein Cytoplasmic side Cytoplasmic vesicle Clathrin-coated vesicle Golgi apparatus Trans-Golgi network Cell projection Ruffle Cytoplasm Localized at sites of endocytosis at the cell membrane. Detected on newly formed macropinosomes. Transiently recruited to clathrin-coated pits at a late stage of clathrin-coated vesicle formation. Colocalizes with the actin cytoskeleton at the cell membrane.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	SNX9: May be involved in several stages of intracellular trafficking. Plays a role in endocytosis via clathrin-coated pits, but also clathrin-independent, actin-dependent fluid-phase endocytosis. Plays a role in macropinocytosis. Promotes internalization of TNFR. Promotes degradation of EGFR after EGF signaling. Stimulates the GTPase activity of DNM1. Promotes DNM1 oligomerization. Promotes activation of the Arp2/3 complex by WASL, and thereby plays a role in the reorganization of the F- actin cytoskeleton. Binds to membranes enriched in phosphatidylinositol 4,5-bisphosphate and promotes membrane tubulation. Has lower affinity for membranes enriched in phosphatidylinositol 3-phosphate. Belongs to the sorting nexin family.
UniProt Protein Details:	Protein type:Vesicle Chromosomal Location of Human Ortholog: 6q25.3 Cellular Component: ruffle; extrinsic to internal side of plasma membrane; cytoplasmic vesicle membrane; clathrin-coated vesicle; cytoplasmic membrane-bound vesicle; cytoplasm; plasma membrane; trans-Golgi network; endosome Molecular Function:protein binding; protein homodimerization activity; ubiquitin protein ligase binding; phosphoinositide binding; phosphatidylinositol binding Biological Process: mitosis; intracellular protein transport; receptor-mediated endocytosis; positive regulation of protein oligomerization; cytokinesis after mitosis; positive regulation of protein kinase activity; vesicle organization and biogenesis; endocytosis; endosome transport; positive regulation of membrane protein ectodomain proteolysis; positive regulation of GTPase activity
NCBI Summary:	This gene encodes a member of the sorting nexin family. Members of this family contain a phosphoinositide binding domain, and are involved in intracellular trafficking. The encoded protein does not contain a coiled coil region, like some family members, but does contain a SRC homology domain near its N-terminus. The encoded protein is reported to have a variety of interaction partners, including of adaptor protein 2 , dynamin, tyrosine kinase non-receptor 2, Wiskott-Aldrich syndrome-like, and ARP3 actin-related protein 3. The encoded protein is implicated in several stages of intracellular trafficking, including endocytosis, macropinocytosis, and F-actin nucleation. [provided by RefSeq, Jul 2013]
UniProt Code:	Q9Y5X1
NCBI GenInfo Identifier:	12643956
NCBI Gene ID:	51429
NCBI Accession:	Q9Y5X1.1
UniProt Secondary Accession:	Q9Y5X1,Q9BSI7, Q9BVM1, Q9UJH6, Q9UP20,
UniProt Related Accession:	Q9Y5X1
Molecular Weight:	595
NCBI Full Name:	Sorting nexin-9
NCBI Synonym Full Names:	sorting nexin 9
NCBI Official Symbol:	SNX9
NCBI Official Synonym Symbols:	SDP1; WISP; SH3PX1; SH3PXD3A
NCBI Protein Information:	sorting nexin-9; SH3 and PX domain-containing protein 1; SH3 and PX domain-containing protein 3A; Wiskott-Aldrich syndrome protein (WASP) interactor protein
UniProt Protein Name:	Sorting nexin-9
UniProt Synonym Protein Names:	SH3 and PX domain-containing protein 1; Protein SDP1; SH3 and PX domain-containing protein 3A
Protein Family:	Sorting nexin
UniProt Gene Name:	SNX9
UniProt Entry Name:	SNX9_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA

Human Sorting nexin-9 / SNX9 ELISA Kit