Rat RAB11A ELISA Kit

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SKU:
RTFI00430
€599

Description

Rat RAB11A ELISA Kit - Information

The Rat RAB11A ELISA Kit can assay for Rat RAB11A in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Rat RAB11A ELISA Kits Work?

The ELISA Genie ELISA (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, We have developed a range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands.

This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with Rat RAB11A. During the reaction, Rat RAB11A in the sample or standard competes with a fixed amount of Rat RAB11A on the solid phase supporter for sites on the Biotinylated Detection Antibody specific to Rat RAB11A. Excess conjugate and unbound sample or standard are washed from the plate, and HRP-Streptavidin(SABC) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Rat RAB11A in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Rat RAB11A ELISA Kit - Data

Description

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.

Post-Translational Modification

Uniprot ID P62494
Detection method

CompetitiveCompetitive

Application

This immunoassay kit allows for the in vitro quantitative determination of Rab11a concentrations in serum plasma and other biological fluids.

Size

96T

Range

1.56-100ng/ml

Sensitivity

< 0.938ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Rab11a and the recovery rates were calculated by comparing the measured value to the expected amount of Rab11a in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 85-96 90
EDTA plasma(n=5) 88-103 93
UFH plasma(n=5) 93-103 98
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rab11a and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 86-102% 93-102% 94-105% 95-101%
EDTA plasma(n=5) 85-100% 86-95% 83-101% 82-97%
UFH plasma(n=5) 82-100% 92-97% 84-97% 80-92%
CV(%)

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

Note

For Research Use Only

Rat RAB11A ELISA Kit Protocol

The below protocol is a sample protocol for a Rat RAB11A ELISA Kit. Competitive ELISA kits allow for the detection and quantification of an analyte in a sample. This Rat RAB11A ELISA Kit allows the researcher to calculate the amount of Rat RAB11A present in their sample. 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.

Competitive ELISA Protocol

ELISA Kit Technical Manual

Kit 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.Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank, or Sample per well. The blank well is added with Sample / Standard dilution buffer. Immediately add 50 µL of Biotin-detection antibody working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming to the best of your ability.)
3.Wash: Aspirate each well and wash, repeating the process three times Wash by filling each well with Wash Buffer (approximately 350µL) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30minutes at 37°C.
5.Wash: Repeat the aspiration/wash process for five times.
6.TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new Plate sealer. Incubate for about 10-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.
7.Stop: Add 50µL of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.
8.OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.

Rat RAB11A ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)60ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Rat RAB11A ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Rat RAB11A ELISA Kit Protein Information

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; G protein, monomeric; G protein, monomeric, Rab; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 8q24

Cellular Component: axon; centrosome; cleavage furrow; cytoplasm; cytoplasmic vesicle; Golgi apparatus; kinetochore microtubule; membrane; mitochondrion; multivesicular body; perinuclear region of cytoplasm; phagocytic vesicle; protein complex; recycling endosome; spindle pole; trans-Golgi network; transport vesicle; vesicle

Molecular Function:GTP binding; GTPase activity; microtubule binding; myosin V binding

Biological Process: astral microtubule organization and biogenesis; establishment of vesicle localization; exocytosis; melanosome transport; mitotic metaphase plate congression; neurite development; positive regulation of axon extension; regulation of long-term neuronal synaptic plasticity; regulation of protein transport; vesicle-mediated transport

UniProt Code:P62494
NCBI GenInfo Identifier:13592148
NCBI Gene ID:81830
NCBI Accession:NP_112414.1
UniProt Secondary Accession:P62494,P24410, Q9JLX1,
Molecular Weight:24,394 Da
NCBI Full Name:ras-related protein Rab-11A
UniProt Protein Name:Ras-related protein Rab-11A
UniProt Synonym Protein Names:24KG
Protein Family:Ras-related protein
UniProt Gene Name:Rab11a  
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Additional Information

Reactivity:
Rat
ELISA Type:
Competitive
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