Rat Mannose Binding Lectin/Mannose Binding Protein ELISA Kit (RTFI00946)

Product Name:	Rat Mannose Binding Lectin/Mannose Binding Protein ELISA Kit
Product Code:	RTFI00946
Size:	96 Assays
Target:	Rat MBP/MBL
Alias:	MBP, MBL, Mbl2, MBL, MBP, MBP1, MBPD, MBL2D, MBP-C, COLEC1, HSMBPC, L-MBP, MBL-C, L-MBP, mannose-binding protein C, mannose-binding lectin, protein C 2
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-100	90
EDTA plasma(n=5)	88-103	96
UFH plasma(n=5)	85-103	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat MBP/MBL 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)	86-103%	88-105%	93-101%
EDTA plasma(n=5)	82-96%	84-101%	87-101%
UFH plasma(n=5)	82-96%	80-99%	80-92%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P02688

UniProt Protein Function:	MBP: The classic group of MBP isoforms (isoform 4-isoform 14) are with PLP the most abundant protein components of the myelin membrane in the CNS. They have a role in both its formation and stabilization. The smaller isoforms might have an important role in remyelination of denuded axons in multiple sclerosis. The non- classic group of MBP isoforms (isoform 1-isoform 3/Golli-MBPs) may preferentially have a role in the early developing brain long before myelination, maybe as components of transcriptional complexes, and may also be involved in signaling pathways in T- cells and neural cells. Differential splicing events combined with optional post-translational modifications give a wide spectrum of isomers, with each of them potentially having a specialized function. Induces T-cell proliferation. Homodimer. Isoform 3 exists as a homodimer. MBP isoforms are found in both the central and the peripheral nervous system, whereas Golli-MBP isoforms are expressed in fetal thymus, spleen and spinal cord, as well as in cell lines derived from the immune system. Belongs to the myelin basic protein family. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cytoskeletal Cellular Component: internode region of axon; compact myelin; cell projection; cell soma; plasma membrane; myelin sheath Molecular Function:protein binding; protease binding; structural constituent of myelin sheath Biological Process: response to mercury ion; myelination; substantia nigra development; response to toxin; negative regulation of axonogenesis; response to progesterone stimulus; aging
NCBI Summary:	The protein encoded by the classic Mbp gene is a major constituent of the myelin sheath of oligodendrocytes and Schwann cells in the nervous system. However, Mbp-related transcripts are also present in the bone marrow and the immune system. These mRNAs arise from the long Mbp gene (otherwise called "Golli-Mbp") that contains 3 additional exons located upstream of the classic Mbp exons. Alternative splicing from the Golli and the Mbp transcription start sites gives rise to 2 sets of Mbp-related transcripts and gene products. The Golli mRNAs contain 3 exons unique to Golli-Mbp, spliced in-frame to 1 or more Mbp exons. They encode hybrid proteins that have N-terminal Golli aa sequence linked to Mbp aa sequence. The second family of transcripts contain only Mbp exons and produce the well characterized myelin basic proteins. This complex gene structure is conserved among species suggesting that the Mbp transcription unit is an integral part of the Golli transcription unit and that this arrangement is important for the function and/or regulation of these genes. Mutation of the Mbp gene is associated with the 'shiverer' and 'myelin deficient' phenotypes in mouse. [provided by RefSeq]
UniProt Code:	P02688
NCBI GenInfo Identifier:	70166270
NCBI Gene ID:	24547
NCBI Accession:	NP_001020465.1
UniProt Secondary Accession:	P02688,Q505J1, Q8R4K6, Q9Z1J4, Q9Z1J5, Q9Z1J6,
UniProt Related Accession:	P02688,Q5XFW1
Molecular Weight:
NCBI Full Name:	myelin basic protein isoform 4
NCBI Synonym Full Names:	myelin basic protein
NCBI Official Symbol:	Mbp
NCBI Official Synonym Symbols:	Mbps
NCBI Protein Information:	Golli-Mbp; myelin basic protein; MBP S; myelin basic protein S; myelin basic protein Golli-mbp; Golli-Mbp; myelin basic protein; myelin basic protein S
UniProt Protein Name:	Myelin basic protein S
Protein Family:	Myelin basic protein
UniProt Gene Name:	Mbp
UniProt Entry Name:	MBP_RAT

Step	Procedure
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.