Human Ras-related protein Rab-10 (RAB10) ELISA Kit

Product Name:	Human Ras-related protein Rab-10 (RAB10) ELISA Kit
SKU:	HUEB2475
Size:	96T
Target:	Human Ras-related protein Rab-10 (RAB10)
Synonyms:	Ras-related protein Rab-10, RAB10
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.073ng/mL

Intra CV:

5.3%

Inter CV:

8.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	110-120%	109-119%	107-117%	110-120%
EDTA Plasma(N=5)	104-113%	109-119%	108-117%	93-105%
Heparin Plasma(N=5)	107-116%	109-120%	107-117%	102-112%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	102	96-108
Plasma	104	98-110

Function:

The small GTPases Rab are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes. Rabs cycle between an inactive GDP-bound form and an active GTP-bound form that is able to recruit to membranes different set of downstream effectors directly responsible for vesicle formation, movement, tethering and fusion (By similarity). That Rab is mainly involved in the biosynthetic transport of proteins from the Golgi to the plasma membrane. Regulates, for instance, SLC2A4/GLUT4 glucose transporter-enriched vesicles delivery to the plasma membrane. In parallel, it regulates the transport of TLR4, a toll-like receptor to the plasma membrane and therefore may be important for innate immune response. Plays also a specific role in asymmetric protein transport to the plasma membrane within the polarized neuron and epithelial cells. In neurons, it is involved in axonogenesis through regulation of vesicular membrane trafficking toward the axonal plasma membrane while in epithelial cells, it regulates transport from the Golgi to the basolateral membrane. Moreover, may play a role in the basolateral recycling pathway and in phagosome maturation. According to PubMed:23263280, may play a role in endoplasmic reticulum dynamics and morphology controlling tubulation along microtubules and tubules fusion.

Uniprot:	P61026
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Ras-related protein Rab-10
Sub Unit:	Interacts with MYO5A; mediates the transport to the plasma membrane of SLC2A4/GLUT4 storage vesicles. Interacts with GDI1 and maybe with GDI2; negatively regulates RAB10 association with membranes and activation. Interacts (GDP-bound form) with LLGL1; the interaction is direct and promotes RAB10 association with membranes and activation through competition with the Rab inhibitor GDI1 (By similarity). Interacts with EXOC4; probably associates with the exocyst (By similarity). Interacts (GTP-bound form) with MICALCL, MICAL1, MICAL3, EHBP1 and EHBP1L1; at least in case of MICAL1 two molecules of RAB10 can bind to one molecule of MICAL1.
Research Area:	Signal Transduction
Subcellular Location:	Cytoplasmic vesicle membrane Lipid-anchor Cytoplasmic side Golgi apparatus membrane Golgi apparatus Trans-Golgi network membrane Endosome membrane Recycling endosome membrane Cytoplasmic vesicle Phagosome membrane Cell projection Cilium Endoplasmic reticulum membrane Associates with SLC2A4/GLUT4 storage vesicles (PubMed:22908308). Localizes to the base of the cilium (PubMed:20576682). Transiently associates with phagosomes (By similarity). Localizes to the endoplasmic reticulum at domains of new tubule growth (PubMed:23263280).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	RAB10: a protein of the GTPase superfamily, Rab family. May be involved in vesicular trafficking and neurotransmitter release. Localized to the inner face of the cell membrane by a lipid-anchor.
UniProt Protein Details:	Protein type:G protein, monomeric; G protein; G protein, monomeric, Rab Chromosomal Location of Human Ortholog: 2p23.3 Cellular Component: Golgi apparatus; phagocytic vesicle membrane; endoplasmic reticulum membrane; recycling endosome; cytoplasmic vesicle membrane; focal adhesion; recycling endosome membrane; exocyst; plasma membrane; endosome membrane; trans-Golgi network; endosome Molecular Function:GTPase activity; GDP binding; GTP binding; myosin V binding Biological Process: metabolic process; establishment of neuroblast polarity; protein secretion; endosome transport; regulation of exocytosis; vesicle-mediated transport; polarized epithelial cell differentiation; intracellular protein transport; Golgi to plasma membrane transport; antigen processing and presentation; cellular response to insulin stimulus; axonogenesis; Golgi to plasma membrane protein transport; Rab protein signal transduction; vesicle docking during exocytosis
NCBI Summary:	RAB10 belongs to the RAS (see HRAS; MIM 190020) superfamily of small GTPases. RAB proteins localize to exocytic and endocytic compartments and regulate intracellular vesicle trafficking (Bao et al., 1998 [PubMed 9918381]).[supplied by OMIM, Mar 2009]
UniProt Code:	P61026
NCBI GenInfo Identifier:	46577638
NCBI Gene ID:	10890
NCBI Accession:	P61026.1
UniProt Related Accession:	P61026
Molecular Weight:	22.5kDa
NCBI Full Name:	Ras-related protein Rab-10
NCBI Synonym Full Names:	RAB10, member RAS oncogene family
NCBI Official Symbol:	RAB10
NCBI Protein Information:	ras-related protein Rab-10
UniProt Protein Name:	Ras-related protein Rab-10
Protein Family:	Ras-related protein
UniProt Gene Name:	RAB10
UniProt Entry Name:	RAB10_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 RAB10 ELISA Kit