Invitrogen

ApoA1 Monoclonal Antibody (513)

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This Antibody was verified by Relative expression to ensure that the antibody binds to the antigen stated. View Details

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ApoA1 Antibody in Immunofluorescence (IF) — Immunofluorescence analysis of Apolipoprotein A1 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Apolipoprotein A1 (513) Mouse Monoclonal Antibody (Product # MIA1404) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

ApoA1 Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry analysis of Apolipoprotein A-1 showing staining in the cytoplasm of paraffin-embedded mouse small intestine tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Apolipoprotein A-1 Mouse Monoclonal Antibody (Product # MIA1404) diluted in 3% BSA-PBS at a dilution of 1:100 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

ApoA1 Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry analysis of Apolipoprotein A-1 showing staining in the cytoplasm of paraffin-embedded human liver tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Apolipoprotein A-1 Mouse Monoclonal Antibody (Product # MIA1404) diluted in 3% BSA-PBS at a dilution of 1:100 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

ApoA1 Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry was performed on human liver tissue sections. To expose Apo A-1, tissues were microwaved for 10 minutes in 10mM sodium citrate, pH 6.0. Following antigen retrieval, endogenous peroxidases were blocked in a 3% hydrogen peroxide-methanol solution for 15 minutes, and then tissues were blocked in 3% BSA in PBS for 30 minutes at room temperature. Tissues were probed with an Apo A-1 monoclonal antibody (Product # MIA1404) at a dilution of 1:50 overnight at 4C, washed extensively in PBST, and detection was performed with an HRP-conjugated anti-mouse IgG secondary antibody followed by DAB substrate. Tissues were counterstained with hematoxylin.

ApoA1 Antibody in Western Blot (WB) — Fig 1: Western blot analysis was performed on tissue extract (30 µg lysate) of Mouse Liver (Lane 1). Fig 2: Likewise, Western blot analysis was performed on samples of 3 µL serum (Lane 1), 5 µL serum (Lane 2), 5 µL plasma (Lane 3), all of which were obtained from a 50 year old male Alzheimer's patient, and 10 µL of conditioned media from HepG2 cell line (lane 4). The blots were probed with Anti-Apolipoprotein A-1 Mouse Monoclonal Antibody (Product # MIA1404, 1:500-1:2000 dilution) and detected by chemiluminescence Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 28 kDa band corresponding to Apolipoprotein A-1 was observed across the cell line, tissues and samples tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

ApoA1 Antibody in Western Blot (WB) — Western blot analysis of Apolipoprotein A-1 was performed by loading 10 µL of serum depleted of highly abundant proteins using the Top 2 Abundant Protein Depletion Spin Columns (Product # 85162, left lane) or the Top 12 Abundant Protein Depletion Spin Columns (Product # 85164, middle lane) and concentrated using the PES Protein Concentrators, 10K MWCO (Product # 88513), 50 µg of HepG2 whole cell lysate (right lane), and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a Nitrocellulose Membrane (Product # 88014) using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. Apo A-1 was detected at ~25 kD using an Apolipoprotein A-1 monoclonal antibody (Product # MIA1404) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG Fc-specific secondary antibody (Product # 31439) at a dilution of 1:40,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

ApoA1 Antibody in Western Blot (WB) — Western blot analysis of Apolipoprotein A-1 was performed by loading the indicated amounts of a recombinant human Apo A-1-hIgG1Fc fusion protein (Product # 10686-H02H-50) or a recombinant mouse Apo A-1-hIgG1Fc fusion protein (Product # 50918-M02H-50), and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a Nitrocellulose Membrane (Product # 88014) using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. Apo A-1 was detected at ~50 kD using an Apolipoprotein A-1 monoclonal antibody (Product # MIA1404) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG Fc-specific secondary antibody (Product # 31439) at a dilution of 1:40,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

ApoA1 Antibody in ELISA (ELISA) — Sandwich ELISA of Apolipoprotein A-1 was performed by coating wells of a 96-well plate with 100 µL of an Apo A-1 Recombinant Rabbit Polyclonal Antibody (Product # 710263) at a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 150 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 30 minutes, and 80 µL of recombinant human Apo A-1 (Product # 10686-H02H-50) standards ranging from 1.6-1000 ng/mL (A) or 100 µL of diluted normal human serum or diluted serum from a patient with dyslipidemia (B) were incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an Apo A-1 Mouse Monoclonal Antibody (Product # MIA1404) was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of a biotinylated Rabbit anti-Mouse IgG Superclonal secondary antibody (Product # A27024) was incubated for 1 hour, followed by Streptavidin-HRP (Product # 21126) for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

ApoA1 Antibody in ELISA (ELISA) — Sandwich ELISA of Apolipoprotein A-1 was performed by coating wells of a 96-well plate with 100 µL of an Apo A-1 rabbit monoclonal antibody (Product # 701239) at a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 150 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 30 minutes, and 80 µL of recombinant human Apo A-1 (Product # 10686-H02H-50) standards ranging from 1.6-1000 ng/mL (A) or 100 µL of diluted normal human serum or diluted serum from a patient with dyslipidemia (B) were incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an Apo A-1 mouse monoclonal antibody (Product # MIA1404) was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of a biotinylated rabbit anti-mouse IgG Superclonal™ secondary antibody (Product # A27024) was incubated for 1 hour, followed by Streptavidin-HRP (Product # 21126) for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

ApoA1 Antibody in ELISA (ELISA) — Sandwich ELISA of Apolipoprotein A-1 was performed by coating wells of a 96-well plate with 100 µL of an Apo A-1 Recombinant Rabbit Polyclonal Antibody (Product # 710263) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 150 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 30 minutes, and 80 µL of recombinant human Apo A-1 (Product # 10686-H02H-50) or recombinant mouse Apo A-1 (Product # 50918-M02H-50) was added to the plate at concentrations ranging from 1.6-1000 ng/mL and incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an Apo A-1 Mouse Monoclonal Antibody (Product # MIA1404) was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of an HRP-conjugated Rabbit anti-Mouse IgG cross-adsorbed secondary antibody (Product # 31452) was incubated for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

ApoA1 Antibody in ELISA (ELISA) — Sandwich ELISA of Apolipoprotein A-1 was performed by coating wells of a 96-well plate with 100 µL of an Apo A-1 rabbit monoclonal antibody (Product # 701239) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 150 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 30 minutes, and 80 µL of recombinant human Apo A-1 (Product # 10686-H02H-50) or recombinant mouse Apo A-1 (Product # 50918-M02H-50) was added to the plate at concentrations ranging from 1.6-1000 ng/mL and incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an Apo A-1 mouse monoclonal antibody (Product # MIA1404) was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of an HRP-conjugated rabbit anti-mouse IgG cross-adsorbed secondary antibody (Product # 31452) was incubated for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

ApoA1 Antibody in Relative expression — Antibody specificity was demonstrated by detection of relative expression of the target protein across patient samples owing to their inherent genetic constitution. Increased expression of Apolipoprotein A1 in serum from patients suffering from dyslipidemia was detected in ELISA using anti-Apolipoprotein A1 monoclonal antibody (513) (Product # MIA1404). {RE}

Product Details

Tested Applications

Dilution

ELISA (ELISA)

1 µg/mL

Immunocytochemistry (ICC)

2 µg/mL

Immunofluorescence (IF)

2 µg/mL

Immunohistochemistry (Paraffin) (IHC (P))

1:100

Immunoprecipitation (IP)

Assay Dependent

Radioimmune Assays (RIA)

Assay Dependent

Western Blot (WB)

1:500-1:2000

Published Applications

Immunohistochemistry (IHC)

See 1 publication below

Product Specifications

Species Reactivity

Human, Mouse

Published species

Human

Host / Isotype

Mouse / IgG1

Class

Monoclonal

Type

Antibody

Clone

513

Immunogen

Purified human plasma Apo A-1

Conjugate

Unconjugated

Form

Liquid

Concentration

3.84 mg/mL

Purification

Ion-exchange chromatography

Storage buffer

0.015M potassium phosphate, pH 7.2, with 0.15M NaCl

Contains

0.1% sodium azide

Storage conditions

Maintain refrigerated at 2-8°C for up to 6 months. For long term storage store at -20°C

RRID

AB_11151929

Product Specific Information

By sandwich ELISA, MIA1404 can be used as a detection antibody with Product # 710263 or # 701239 as a coating antibody, to generate a matched pair. Using these matched pairs, recombinant human Apo A-1, but not recombinant mouse Apo A-1, was detected. MIA1404 can be used to detect Apo A-1 from serum samples. To increase sensitivity of sandwich ELISAs with MIA1404, a biotinylated detection antibody followed by Streptavidin-HRP is recommended.

By Western blot, MIA1404 detects recombinant human Apo A-1, but not recombinant mouse Apo A-1. MIA1404 is also recommended for detecting endogenous Apo A-1 by Western blot.

MIA1404 was formerly sold as a Seradyn product.

Target Information

This gene encodes apolipoprotein A-I, which is the major protein component of high density lipoprotein (HDL) in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion, and it is a cofactor for lecithin cholesterolacyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters. This gene is closely linked with two other apolipoprotein genes on chromosome 11. Defects in this gene are associated with HDL deficiencies, including Tangier disease, and with systemic non-neuropathic amyloidosis.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: Apo-AI; apoA-I; Apolipoprotein A-I; Apolipoprotein A-I(1-242); Apolipoprotein A1; ProapoA-I; Proapolipoprotein A-I; Truncated apolipoprotein A-I

Gene Aliases: Alp-1; apo(a); apo-AI; Apoa-1; apoA-I; APOA1; Brp-14; Ltw-1; Lvtw-1; Sep-1; Sep-2; Sep2

UniProt ID: (Human) P02647, (Mouse) Q00623

Entrez Gene ID: (Human) 335, (Mouse) 11806

regulation of protein phosphorylation endothelial cell proliferation negative regulation of cytokine secretion involved in immune response lipid metabolic process phospholipid metabolic process phosphatidylcholine biosynthetic process cholesterol biosynthetic process transport lipid transport G-protein coupled receptor signaling pathway integrin-mediated signaling pathway protein localization steroid metabolic process cholesterol metabolic process glucocorticoid metabolic process negative regulation of tumor necrosis factor-mediated signaling pathway positive regulation of cholesterol esterification positive regulation of triglyceride catabolic process negative regulation of very-low-density lipoprotein particle remodeling phospholipid transport protein oxidation peptidyl-methionine modification triglyceride catabolic process lipid storage regulation of intestinal cholesterol absorption cholesterol transport adrenal gland development organ regeneration axon regeneration regulation of Cdc42 protein signal transduction cholesterol efflux phospholipid efflux negative regulation of heterotypic cell-cell adhesion very-low-density lipoprotein particle remodeling high-density lipoprotein particle remodeling high-density lipoprotein particle assembly positive regulation of Rho protein signal transduction lipoprotein metabolic process lipoprotein biosynthetic process cholesterol homeostasis blood vessel endothelial cell migration reverse cholesterol transport positive regulation of fatty acid biosynthetic process phosphatidylcholine metabolic process negative regulation of interleukin-1 beta secretion negative regulation of inflammatory response protein stabilization negative chemotaxis positive regulation of lipoprotein lipase activity vitamin transport positive regulation of hydrolase activity negative regulation of hydrolase activity positive regulation of transferase activity positive regulation of stress fiber assembly phospholipid homeostasis negative regulation of lipase activity negative regulation of cell adhesion molecule production negative regulation of response to cytokine stimulus triglyceride homeostasis cholesterol import positive regulation of substrate adhesion-dependent cell spreading retinoid metabolic process platelet degranulation receptor-mediated endocytosis transforming growth factor beta receptor signaling pathway response to nutrient peripheral nervous system axon regeneration neuron projection regeneration high-density lipoprotein particle clearance response to drug response to estrogen cellular protein metabolic process transmembrane transport ERK1 and ERK2 cascade

Process(es)

beta-amyloid binding protein binding phospholipid binding phospholipid transporter activity high-density lipoprotein particle binding cholesterol binding cholesterol transporter activity enzyme binding phosphatidylcholine binding apolipoprotein receptor binding apolipoprotein A-I receptor binding identical protein binding chemorepellent activity lipase inhibitor activity phosphatidylcholine-sterol O-acyltransferase activator activity high-density lipoprotein particle receptor binding lipid transporter activity lipid binding lipoprotein particle binding