Product Name:	Mouse ApoB / Apolipoprotein B ELISA Kit
Product Code:	MOFI00644
Size:	96 Assays
Alias:	ApoB, Apolipoprotein B, APOB, Apo-B, FLDB, LDLCQ4
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse ApoB concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ug/ml
Range:	0.313-20ug/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-105	92
EDTA plasma(n=5)	86-97	90
UFH plasma(n=5)	86-102	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse ApoB 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)	86-101%	85-100%	92-105%
EDTA plasma(n=5)	87-100%	83-99%	86-98%
UFH plasma(n=5)	91-99%	84-93%	80-91%

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

E9Q414

UniProt Protein Function:	APOB: Apolipoprotein B is a major protein constituent of chylomicrons (apo B-48), LDL (apo B-100) and VLDL (apo B-100). Apo B-100 functions as a recognition signal for the cellular binding and internalization of LDL particles by the apoB/E receptor. Defects in APOB are a cause of familial hypobetalipoproteinemia type 1 (FHBL1). A disorder characterized by highly reduced plasma concentrations of low density lipoproteins, and dietary fat malabsorption. Clinical presentation may vary from no symptoms to severe gastrointestinal and neurological dysfunction similar to abetalipoproteinemia. Defects in APOB are a cause of familial ligand-defective apolipoprotein B-100 (FDB). FDB is a dominantly inherited disorder of lipoprotein metabolism leading to hypercholesterolemia and increased proneness to coronary artery disease (CAD). The plasma cholesterol levels are dramatically elevated due to impaired clearance of LDL particles by defective APOB/E receptors. Defects in APOB associated with defects in other genes (polygenic) can contribute to hypocholesterolemia.
UniProt Protein Details:	Protein type:Carrier; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 12 A1.1\|12 3.53 cM Cellular Component: cell soma; chylomicron; cytoplasm; cytosol; endoplasmic reticulum; endoplasmic reticulum exit site; extracellular exosome; extracellular region; extracellular space; intermediate-density lipoprotein particle; intracellular membrane-bound organelle; low-density lipoprotein particle; mature chylomicron; very-low-density lipoprotein particle; vesicle membrane Molecular Function:cholesterol transporter activity; heparin binding; lipid binding; lipid transporter activity; low-density lipoprotein receptor binding; phospholipid binding Biological Process: artery morphogenesis; cholesterol efflux; cholesterol homeostasis; cholesterol metabolic process; cholesterol transport; fertilization; in utero embryonic development; lipid catabolic process; lipid metabolic process; lipid transport; lipoprotein biosynthetic process; lipoprotein catabolic process; lipoprotein metabolic process; lipoprotein transport; low-density lipoprotein particle clearance; low-density lipoprotein particle remodeling; nervous system development; positive regulation of cholesterol storage; positive regulation of gene expression; positive regulation of lipid storage; positive regulation of macrophage derived foam cell differentiation; post-embryonic development; regulation of cholesterol biosynthetic process; response to carbohydrate stimulus; sperm motility; spermatogenesis; steroid metabolic process; transport; triacylglycerol mobilization; triglyceride catabolic process
NCBI Summary:	This gene product is the main apolipoprotein of chylomicrons and low density lipoproteins. It occurs in plasma as two main isoforms, apoB-48 and apoB-100. Unlike the apoB-48 and apoB-100 structural equivalents in human, which are synthesized exclusively in the gut and liver, respectively, the mouse apoB-48 isoform is also found in mouse liver. The intestinal and the hepatic forms of apoB are encoded by a single gene from a single, very long mRNA. The two isoforms share a common N-terminal sequence. The shorter apoB-48 protein is produced after RNA editing of the apoB-100 transcript at residue 2179 (CAA->UAA), resulting in the creation of a stop codon, and early translation termination. [provided by RefSeq, Jul 2008]
UniProt Code:	E9Q414
NCBI GenInfo Identifier:	161702988
NCBI Gene ID:	238055
NCBI Accession:	NP_033823.2
UniProt Secondary Accession:	E9Q414,Q3UH74, Q497D8, Q61314, Q61318, Q8CGG8,
UniProt Related Accession:	E9Q414
Molecular Weight:
NCBI Full Name:	apolipoprotein B-100
NCBI Synonym Full Names:	apolipoprotein B
NCBI Official Symbol:	Apob
NCBI Official Synonym Symbols:	apob-48; AI315052; apob-100; Apo B-100
NCBI Protein Information:	apolipoprotein B-100
UniProt Protein Name:	Apolipoprotein B-100
Protein Family:	Apolipoprotein
UniProt Gene Name:	Apob

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

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 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.