Human RAB11A ELISA Kit (HUEB1897)- High Sensitivity

Product Name:	Human Ras-related protein Rab-11A (RAB11A) ELISA Kit
SKU:	HUEB1897
Size:	96T
Target:	Human Ras-related protein Rab-11A (RAB11A)
Synonyms:	YL8, Rab-11, RAB11
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.78-50ng/mL
Sensitivity:	0.34ng/mL

Intra CV:

5.2%

Inter CV:

8.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	96-106%	107-117%	107-117%	108-116%
EDTA Plasma(N=5)	99-109%	115-125%	88-100%	98-108%
Heparin Plasma(N=5)	103-113%	82-92%	109-120%	99-109%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	97	91-103
Plasma	99	93-105

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. That Rab regulates endocytic recycling. Acts as a major regulator of membrane delivery during cytokinesis. Together with MYO5B and RAB8A participates in epithelial cell polarization. Together with RAB3IP, RAB8A, the exocyst complex, PARD3, PRKCI, ANXA2, CDC42 and DNMBP promotes transcytosis of PODXL to the apical membrane initiation sites (AMIS), apical surface formation and lumenogenesis. Together with MYO5B participates in CFTR trafficking to the plasma membrane and TF (Transferrin) recycling in nonpolarized cells. Required in a complex with MYO5B and RAB11FIP2 for the transport of NPC1L1 to the plasma membrane. Participates in the sorting and basolateral transport of CDH1 from the Golgi apparatus to the plasma membrane. Regulates the recycling of FCGRT (receptor of Fc region of monomeric Ig G) to basolateral membranes. May also play a role in melanosome transport and release from melanocytes.

Uniprot:	P62491
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Ras-related protein Rab-11A
Sub Unit:	Interacts with RIP11 and STXBP6. Interacts with SGSM1, SGSM2 and SGSM3 (By similarity). Interacts with EXOC6 in a GTP-dependent manner (By similarity). Interacts with RAB11FIP1, RAB11FIP2, RAB11FIP3 (via its C-terminus) and RAB11FIP4. Interacts with EVI5; EVI5 and RAB11FIP3 may be mutually exclusive and compete for binding RAB11A. Interacts with VIPAS39. Interacts with MYO5B. Found in a complex with MYO5B and CFTR. Interacts with NPC1L1. Interacts (GDP-bound form) with ZFYVE27 (PubMed:21976701, PubMed:17082457). Interacts with BIRC6/bruce. May interact with TBC1D14. Interacts with UNC119; in a cell cycle-dependent manner. GDP-bound and nucleotide-free forms interact with SH3BP5 (PubMed:26506309). Interacts (GDP-bound form) with KIF5A in a ZFYVE27-dependent manner (PubMed:21976701).
Subcellular Location:	Cell membrane Lipid-anchor Recycling endosome membrane Lipid-anchor Cleavage furrow Cytoplasmic vesicle Phagosome Translocates with RAB11FIP2 from the vesicles of the endocytic recycling compartment (ERC) to the plasma membrane (PubMed:11994279). Localizes to the cleavage furrow (PubMed:15601896). Colocalizes with PARD3, PRKCI, EXOC5, OCLN, PODXL and RAB8A in apical membrane initiation sites (AMIS) during the generation of apical surface and lumenogenesis (PubMed:20890297). Recruited to phagosomes containing S.aureus or M.tuberculosis (PubMed:21255211).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	RAB11A: Regulates endocytic recycling. May exert its functions by interacting with multiple effector proteins in different complexes. Acts as a major regulator of membrane delivery during cytokinesis. Together with MYO5B and RAB8A participates in epithelial cell polarization. Together with RAB3IP, RAB8A, the exocyst complex, PARD3, PRKCI, ANXA2, CDC42 and DNMBP promotes transcytosis of PODXL to the apical membrane initiation sites (AMIS), apical surface formation and lumenogenesis. Together with MYO5B participates in CFTR trafficking to the plasma membrane and TF (Transferrin) recycling in nonpolarized cells. Required in a complex with MYO5B and RAB11FIP2 for the transport of NPC1L1 to the plasma membrane. Participates in the sorting and basolateral transport of CDH1 from the Golgi apparatus to the plasma membrane. Regulates the recycling of FCGRT (receptor of Fc region of monomeric Ig G) to basolateral membranes. Interacts with RIP11 and STXBP6. Interacts with SGSM1, SGSM2 and SGSM3. Interacts with EXOC6 in a GTP- dependent manner. Interacts with RAB11FIP1, RAB11FIP2, RAB11FIP3 (via its C-terminus) and RAB11FIP4. Interacts with EVI5; EVI5 and RAB11FIP3 may be mutually exclusive and compete for binding RAB11A. Interacts with SPE39//C14orf133. Interacts with MYO5B. Found in a complex with MYO5B and CFTR. Interacts with NPC1L1. Interacts (GDP-bound form) with ZFYVE27. Interacts with BIRC6/bruce. Belongs to the small GTPase superfamily. Rab family.
UniProt Protein Details:	Protein type:G protein, monomeric; G protein; G protein, monomeric, Rab; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 15q22.31 Cellular Component: Golgi apparatus; spindle pole; protein complex; cytoplasmic vesicle membrane; mitochondrion; recycling endosome membrane; phagocytic vesicle; trans-Golgi network; cytosol; kinetochore microtubule; multivesicular body; recycling endosome; phagocytic vesicle membrane; transport vesicle; perinuclear region of cytoplasm; axon; plasma membrane; cytoplasmic vesicle; vesicle; cleavage furrow Molecular Function:GTPase activity; protein binding; GDP binding; syntaxin binding; GTP binding; microtubule binding; myosin V binding Biological Process: regulation of long-term neuronal synaptic plasticity; establishment of vesicle localization; exocytosis; metabolic process; organelle organization and biogenesis; positive regulation of axon extension; water transport; cytokinesis; melanosome transport; mitotic metaphase plate congression; vesicle-mediated transport; regulation of protein transport; intracellular protein transport; astral microtubule organization and biogenesis; renal water homeostasis; plasma membrane to endosome transport; Rab protein signal transduction; transmembrane transport; neurite development
NCBI Summary:	The protein encoded by this gene belongs to the Rab family of the small GTPase superfamily. It is associated with both constitutive and regulated secretory pathways, and may be involved in protein transport. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2011]
UniProt Code:	P62491
NCBI GenInfo Identifier:	50402542
NCBI Gene ID:	8766
NCBI Accession:	P62491.3
UniProt Secondary Accession:	P62491,P24410, Q5TZN9, Q9JLX1, B2R4B6, B4DT13,
UniProt Related Accession:	P62491
Molecular Weight:	17,659 Da
NCBI Full Name:	Ras-related protein Rab-11A
NCBI Synonym Full Names:	RAB11A, member RAS oncogene family
NCBI Official Symbol:	RAB11A
NCBI Official Synonym Symbols:	YL8
NCBI Protein Information:	ras-related protein Rab-11A; rab-11; RAB 11A, member oncogene family
UniProt Protein Name:	Ras-related protein Rab-11A
UniProt Synonym Protein Names:	YL8
Protein Family:	Ras-related protein
UniProt Gene Name:	RAB11A
UniProt Entry Name:	RB11A_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 RAB11A ELISA Kit