- MIA1404 - Invitrogen Antibodies APOA1 antibody

Submitted references Selective activation of ABCA1/ApoA1 signaling in the V1 by magnetoelectric stimulation ameliorates depression via regulation of synaptic plasticity.

Lu Q, Wu F, Jiao J, Xue L, Song R, Shi Y, Kong Y, Sun J, Gu N, Han MH, Zhang Z
iScience 2022 May 20;25(5):104201

Apolipoprotein A-I and B distribution in the human cornea.

Ashraf F, Cogan DG, Kruth HS
Investigative ophthalmology & visual science 1993 Dec;34(13):3574-8

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Apolipoprotein A-1 was done on Hep G2 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Apolipoprotein A-1 Mouse Monoclonal Antibody (MIA1404, red histogram) or with mouse isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Activated ABAC1/ApoA1 signaling is involved in the antidepressant action of c-MSST targeting the left V1 of CUMS mice (A and B) Heat map of the differentially expressed proteins analyzed by iTRAQ (Group A, Control mice; Group B, CUMS mice; Group C, Sham c-MSST mice; Group D, c-MSST mice), n = 3 mice per group. (C and D) PPI network of the differential proteins in the four groups. The red circles indicate the major/hub nodes of the PPI network. (E and F) Representative fluorescence images of the mouse V1. Immunofluorescent double labeling for ApoA1 (red)/NeuN (green) and ABCA1 (green)/NeuN (red). Nuclei were stained blue with DAPI. n = 4 mice per group. Scale bar, 20 mum. (G) Colocalization of the three neurocytemarkers with ApoA1 was quantified in the left V1 of control and CUMS mice. n = 8-9 mice per group. Data are expressed as the mean +-SEM. ***p < 0.001 versus Con-NeuN+/ApoA1+; ### p < 0.001 versus CUMS-NeuN+/ApoA1+ using ANOVA with Sidak correction. (H-J) Expression of ApoA1 and ABCA1 in the left V1 of control and CUMS mice. n = 6 mice per group. Data are expressed as mean +-SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using ANOVA with Bonferroni correction. iTRAQ, isobaric tagging for relative and absolute quantification; PPI, protein-protein interaction; ApoA1, apolipoprotein A1; ABCA1, ATP-binding cassette, subfamily A, member one; NeuN, neuronal nuclei; DAPI, 4',6-diamidino-2-phenylindole; SPIO, superparamagnetic iron oxide; MF, magnetic field; c-MSST, combined magnetic

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Neuron-specific knockdown of ABCA1 blocks the antidepressant effects of c-MSST via regulation of synaptic plasticity in the mouse V1 region (A) Representative immunoblots and bar graphs showing the expression of ApoA1, PSD-95, synapsin-1 and SYN in the mouse V1 after 5 days of 5 Hz c-MSST. n = four to six mice per group. Data are expressed as mean +-SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus AAV-Con group, # p < 0.05, ### p < 0.001 using ANOVA with Bonferroni correction. (B) TBS stimulation parameter. (C) Representative traces were taken at time points 1 and 2 indicated in the summary plots; fEPSPs recorded for each condition before (1) and 45 min after the application of TBS (2). (D and E) Averaged fEPSP amplitude and summary plots of LTP induced by TBS. n = 7-13 mice per group. Data are expressed as mean +- SEM *p < 0.05, ***p < 0.001 using ANOVA with Bonferroni correction. c-MSST, combined magnetic stimulation system treatment; CUMS, chronic unpredictable mild stress; ApoA1, apolipoprotein A1; PSD-95, postsynaptic density-95; SYN, synaptophysin; fEPSP, field excitatory postsynaptic potentiates; TBS, theta-burst stimulation; LTP, long-term potentiation. See also Figures S6 and S7 .

MIA1404

Antibody data