Human RAB8A/Ras-related protein Rab-8A ELISA Kit (HUFI01328)

Product Name:	Human RAB8A/Ras-related protein Rab-8A ELISA Kit
Product Code:	HUFI01328
Size:	96 Assays
Alias:	RAB8A, Ras-related protein Rab-8A, Oncogene c-mel, MEL, RAB8
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human RAB8A 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 RAB8A and the recovery rates were calculated by comparing the measured value to the expected amount of Human RAB8A in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	93-105	99
EDTA plasma(n=5)	87-99	94
UFH plasma(n=5)	88-102	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human RAB8A 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)	86-104%	92-102%	91-103%
EDTA plasma(n=5)	83-90%	85-99%	86-96%
UFH plasma(n=5)	85-91%	93-100%	80-95%

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

P61006

UniProt Protein Function:	RAB8A: May be involved in vesicular trafficking and neurotransmitter release. Together with RAB11A, RAB3IP, 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 and RAB11A participates in epithelial cell polarization. Interacts with MAP4K2 and SYTL4. Interacts with SGSM1 and SGSM3. Interacts with OPTN. Interacts with RAB3IP. Interacts with MYO5B. Interacts with PIFO/C1orf88. Interacts with BIRC6/bruce. Interacts with OCRL. Belongs to the small GTPase superfamily. Rab family.
UniProt Protein Details:	Protein type:G protein; Motility/polarity/chemotaxis; G protein, monomeric, Rab; G protein, monomeric; Oncoprotein Chromosomal Location of Human Ortholog: 19p13.1 Cellular Component: centrosome; cytoplasmic vesicle membrane; secretory granule membrane; recycling endosome membrane; nonmotile primary cilium; dendrite; postsynaptic density; phagocytic vesicle; cytosol; cilium; nucleoplasm; Golgi membrane; synaptic vesicle; trans-Golgi network transport vesicle; phagocytic vesicle membrane; cell soma; plasma membrane; nucleolus; nucleus Molecular Function:GTPase activity; protein binding; GDP binding; GTP binding; myosin V binding; protein kinase binding; Rab GTPase binding Biological Process: regulation of long-term neuronal synaptic plasticity; metabolic process; organelle organization and biogenesis; protein secretion; regulation of protein transport; regulation of exocytosis; intracellular protein transport; synaptic vesicle exocytosis; axonogenesis; Golgi vesicle fusion to target membrane; cellular response to insulin stimulus; cilium biogenesis; mitotic cell cycle; Rab protein signal transduction; G2/M transition of mitotic cell cycle; vesicle docking during exocytosis
NCBI Summary:	The protein encoded by this gene is a member of the RAS superfamily which are small GTP/GDP-binding proteins with an average size of 200 amino acids. The RAS-related proteins of the RAB/YPT family may play a role in the transport of proteins from the endoplasmic reticulum to the Golgi and the plasma membrane. This protein shares 97%, 96%, and 51% similarity with the dog RAB8, mouse MEL, and mouse YPT1 proteins, respectively and contains the 4 GTP/GDP-binding sites that are present in all the RAS proteins. The putative effector-binding site of this protein is similar to that of the RAB/YPT proteins. However, this protein contains a C-terminal CAAX motif that is characteristic of many RAS superfamily members but which is not found in YPT1 and the majority of RAB proteins. Although this gene was isolated as a transforming gene from a melanoma cell line, no linkage between MEL and malignant melanoma has been demonstrable. This oncogene is located 800 kb distal to MY09B on chromosome 19p13.1. [provided by RefSeq, Jul 2008]
UniProt Code:	P61006
NCBI GenInfo Identifier:	46810392
NCBI Gene ID:	4218
NCBI Accession:	P61006.1
UniProt Secondary Accession:	P61006,P24407, Q6FHV5, B4DEK7,
UniProt Related Accession:	P61006
Molecular Weight:	207
NCBI Full Name:	Ras-related protein Rab-8A
NCBI Synonym Full Names:	RAB8A, member RAS oncogene family
NCBI Official Symbol:	RAB8A
NCBI Official Synonym Symbols:	MEL; RAB8
NCBI Protein Information:	ras-related protein Rab-8A; oncogene c-mel; ras-associated protein RAB8; mel transforming oncogene (RAB8 homolog); mel transforming oncogene (derived from cell line NK14); mel transforming oncogene (derived from cell line NK14)- RAB8 homolog
UniProt Protein Name:	Ras-related protein Rab-8A
UniProt Synonym Protein Names:	Oncogene c-mel
UniProt Gene Name:	RAB8A
UniProt Entry Name:	RAB8A_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.

Human RAB8A/Ras-related protein Rab-8A ELISA Kit (HUFI01328)

Description