MA1-516

antibody from Invitrogen Antibodies

Targeting: HIF1A bHLHe78, HIF-1alpha, HIF1, MOP1, PASD8

Provider product page for MA1-516

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Gel shift

Other assay

Antibody data

Antibody Data
Antigen structure
References [31]
Comments [0]
Validations
Immunocytochemistry [6]
Immunohistochemistry [1]
Other assay [9]

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Product number: MA1-516 - Provider product page
Provider: Invitrogen Antibodies
Product name: HIF1A Monoclonal Antibody (mgc3)
Antibody type: Monoclonal
Antigen: Other
Description: MA1-516 detects hypoxia-inducible factor 1 alpha (HIF-1 alpha) from human, non-human primate, bovine, mouse and porcine cells. This antibody does not cross-react with ARNT or the related HIF-2 alpha. MA1-516 has been successfully used in Western blot, immunofluorescence, immunoprecipitation, immunocytochemistry, and gel shift procedures. By Western blot, this antibody detects an ~116 kDa protein representing HIF-1 alpha after hypoxic induction in COS cells. Immunofluorescence staining of HIF-1 alpha in COS-7 cells with MA1-516 yields nuclear staining after exposing cells to 1% oxygen for 4 hours. In gel shift assays, MA1-516 has been successfully used only with mouse HIF-1 alpha. Antibodies to this protein (and modification) were previously sold as part of a Thermo Scientific Cellomics High Content Screening Kit. This replacement antibody is now recommended for researchers who need an antibody for high content cell based assays. It has been thoroughly tested and validated for cellular immunofluorescence (IF) applications. Further optimization including the selection of the most appropriate fluorescent Dylight conjugated secondary antibody may have to be performed for your high content assay.
Reactivity: Human, Mouse, Bovine, Porcine
Host: Mouse
Isotype: IgG
Antibody clone number: mgc3
Vial size: 100 μL
Concentration: 1 mg/mL
Storage: -20°C, Avoid Freeze/Thaw Cycles

HIF1A protein structure - MA1-516 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HIF-1 alpha (green) in HeLa cells either left untreated (left panel) or treated with 100uM Deferoxamine mesylate for ~16 hours (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a HIF-1 alpha monoclonal antibody (Product # MA1-516) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:500 for 30 minutes at room temperature. F-actin (red) was stained with DyLight 594 Phalloidin (Product # 21836) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HIF-1 alpha (green) in A549 cells either left untreated (left panel) or treated with 100uM desferrioxamine (DFX) for ~16 hours (right panel). The cells were fixed with 4% Paraformaldehyde for 15 min at room temp, permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a HIF-1 alpha monoclonal antibody (Product # MA1-516) at a dilution of 1:100 overnight at 4C, washed with PBS, and incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Product # R37120) at a dilution of 1:500 for 2 hours at room temperature. F-actin (red) was stained with Alexa Fluor 647 Phalloidin (Product # A22287) and nuclei (blue) were stained with DAPI (Product # P-36931). Images were taken on a Zeiss LSM 710 confocal microscope.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence staining of HIF-1 Alpha using Product # MA1-516.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence staining of HIF-1 Alpha using Product # MA1-516.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HIF-1 alpha (green) in HeLa cells either left untreated (left panel) or treated with 100uM Deferoxamine mesylate for ~16 hours (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a HIF-1 alpha monoclonal antibody (Product # MA1-516) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:500 for 30 minutes at room temperature. F-actin (red) was stained with DyLight 594 Phalloidin (Product # 21836) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HIF-1 alpha (green) in A549 cells either left untreated (left panel) or treated with 100uM desferrioxamine (DFX) for ~16 hours (right panel). The cells were fixed with 4% Paraformaldehyde for 15 min at room temp, permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a HIF-1 alpha monoclonal antibody (Product # MA1-516) at a dilution of 1:100 overnight at 4C, washed with PBS, and incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Product # R37120) at a dilution of 1:500 for 2 hours at room temperature. F-actin (red) was stained with Alexa Fluor 647 Phalloidin (Product # A22287) and nuclei (blue) were stained with DAPI (Product # P-36931). Images were taken on a Zeiss LSM 710 confocal microscope.

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Microglial gene expression and function 6 months after immune stimulation. a, Weighted gene correlation network analysis (top: correlation coefficient; bottom: P-value; n=9,9,6,6,5,4 animals). b , Selected KEGG pathways enriched in modules. c , Heatmaps of genes within modules, z-scores (boxplot whiskers: 5-95 th percentile; n=1601,990,949,3543 genes in modules) and selected genes. d , Microglial mitochondrial membrane potential (left/middle; n=9,6,6,8,3,4 animals) and Pearson's correlation with lactate release (right; n=11,10,10 animals). e , Staining for top : HIF-1alpha, microglia (CD11b) and amyloid plaques (Methoxy-X04) and bottom : HIF-1alpha and microglial nuclei (Pu.1; single confocal plane) in brain sections from 9-month-old animals. Scale bars: 20/5 um (top/bottom). f , Total cellular (n=7,7,7 animals) and nuclear (n=8,8,7 animals) HIF-1alpha staining intensity. g , Microglial Abeta content (n=5,11,10,10 animals). */**/*** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Effect of HIF-1alpha siRNA on HIF-1alpha protein expression. Cells were transfected with 15 and 25 nM HIF-1alpha siRNA and an siRNA non targeting control as indicated for 6 h. Twenty-four hours after transfection, cells were exposed to 4 h of hypoxia. Protein was collected immediately after treatment and blotted for HIF-1alpha. Bar graph represents the band intensity relative to the control. Error bars represent the standard error of the mean (n=3). beta-actin is detected as loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 The expression levels of HIF-1alpha were detected by immunoblot analysis in a time-course experiment performed on MDA-MB 468, HepG2, LNCaP, and MCF-7 cells treated with beta-HCH (10 muM). Actin was used as normalization protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 Immunoblot analysis of HIF-1alpha expression in MDA-MB 468, HepG2, LNCaP, and MCF-7 cells after 8 h treatment with 10 muM beta-HCH. Analysis was performed after 8 h treatment with 10 muM beta-HCH, as well as on cells pre-incubated with a specific STAT3 inhibitor (S3I-201) before beta-HCH treatment. Actin was used as normalization protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Effect of Cadmium on HIF-1alpha accumulation and hypoxia preconditioning protection. A : Cells were incubated under normoxia with 200 muM CoCl 2 (as a positive control), 5 muM CdCl 2 alone, hypoxia (0.5% O 2 ) alone, or hypoxia with CdCl 2 for 4 h. Whole cell lysates were collected immediately after treatment, separated on SDS-PAGE gel and blotted for HIF-1alpha. beta-actin was detected as a loading control. B : Cells were pretreated with hypoxia with or without CdCl 2 for 4 h and irradiated 2 min with UV. Four hours after irradiation, cells were fixed and stained for TUNEL. Bar graph shows percentage of TUNEL positive cells in indicated groups. Error bars represent standard error of the mean (n=3); the asterisk indicates significantly different from UV control (p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Effect of HIF-1alpha siRNA on hypoxia-induced VEGF expression. Bovine stromal cells were transfected with HIF-1alpha siRNA or non-targeting siRNA control for 6 h. Twenty-four hours after transfection cells were preconditioned with hypoxia for 4 h. A : Total RNA was collected immediately after treatment. Image shows RT-PCR analysis for HIF-1alpha and VEGF . 18s rRNA was detected as an internal control. B : Protein analysis of VEGF and HIF-1alpha. Whole cell lysates were collected immediately after treatment, separated by SDS PAGE and probed for VEGF and HIF-1alpha. beta-actin was detected as a loading control. Bar graph shows VEGF expression in indicated groups relative to control. Error bars represent standard error of the mean (n=3).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. Hypoxia controls Gal-1 expression in KS through HIF-independent, NF-kappaB-dependent mechanisms. (A-D) Expression of Gal-1 in KS cells transfected with or without HIF-1alpha siRNA or a super-repressor form of IkappaB-alpha (IkappaB-alpha-SR) and incubated under hypoxia or normoxia. (A) Promoter activity. (B) qRT-PCR of Gal-1 mRNA relative to RN18S1. AU, arbitrary units. Data are the mean +- SEM of five (A) or three (B) independent experiments. (C) Immunoblot of Gal-1, IkappaB-alpha, HIF-1alpha, and actin. Data are representative of four independent experiments. (D) ELISA of Gal-1 secretion. Data are the mean +- SEM of three independent experiments. (E) ELISA of Gal-1 secretion by KS cells cultured under hypoxic or normoxic conditions in the presence or absence of HIF-1alpha or NF-kappaB inhibitors. Data are the mean +- SEM of three independent experiments. (F and G) Immunoblot (F) and qRT-PCR (G) of Gal-1 expression induced by hypoxia (Hyp) in human and mouse melanoma (A375 and B16-F0), mouse breast carcinoma (4T1), and human prostate carcinoma (LNCaP) cell lines. Data are representative (F) or are the mean +- SEM (G) of three independent experiments. (H) Western blot of HIF-1alpha, IkappaB-alpha, Gal-1, and actin upon treatment of KS cells with CoCl 2 (chemical activator of HIF-1alpha). Data are representative of four experiments. (I) Gal-1 promoter activity upon treatment of KS cells with CoCl 2 . Modulation of pGL3-Gal-1-Luciferase activity relative to Renilla ex

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 Arginine inhibits HIF-1alpha to reduce LDHA expression after cerebral I/R injury. a Western blotting shows that HIF-1alpha and LDHA are raised in MCAO rats and the increase is attenuated by arginine administration (n = 6 in each group, *p < 0.05 versus sham, # p < 0.05 versus vehicle, two-way ANOVA test). b Western blotting shows that HIF-1alpha and LDHA are raised in in OGD microglia and the increase is attenuated by arginine administration (n = 6 in each group, *p < 0.05 versus sham, # p < 0.05 versus vehicle, two-way ANOVA test). c Western blotting shows that administration of the HIF-1alpha inhibitor, LW6 decreases the protein level of LDHA in MCAO rats and occludes the effect of arginine (n = 6 in each group, *p < 0.05 versus vehicle, one-way ANOVA test). d Western blotting shows that administration of LW6 decreases the level of LDHA in microglia and occludes the effect of arginine (n = 6 in each group, *p < 0.05 versus OGD, one-way ANOVA test). e Western blotting shows that transfection of WT-HIF-1alpha abolishes arginine-induced LDHA reduction in OGD BV-2 cells (n = 6 in each group, *p < 0.05 versus OGD, one-way ANOVA test)