MA3-060

antibody from Invitrogen Antibodies

Targeting: ARF6

Provider product page for MA3-060

Western blot

Immunoprecipitation

Antibody data

Antibody Data
Antigen structure
References [0]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [4]
Immunohistochemistry [3]
Flow cytometry [3]
Other assay [1]

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Product number: MA3-060 - Provider product page
Provider: Invitrogen Antibodies
Product name: ARF1/ARF3/ARF5/ARF6 Monoclonal Antibody (1D9)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA3-060 detects ADP-ribosylation factor1 (Arf1), Arf3, Arf5 and Arf6 and about ten-fold less well with Arf4 from human, mouse and rat tissues. MA3-060 has been successfully used in Western blot, immunofluorescence, immunocytochemistry, and immunoprecipitation procedures. By Western blot, this antibody detects a single ~21 kDa protein representing Arf in rat pancreas extract. Immunofluorescence staining of Arf in rat pancreas with MA3-060 results in staining of the cytoplasmic face of the trans-Golgi membranes. MA3-060 cannot be used to neutralize Arf in solution. The MA3-060 antigen is recombinant human Arf1.
Reactivity: Human, Mouse, Rat, Canine, Hamster
Host: Mouse
Isotype: IgG
Antibody clone number: 1D9
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

ARF6 protein structure - MA3-060 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Arf on canine heart extract using Product # MA3-060.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Arf on canine heart extract using Product # MA3-060.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), MCF7 (Lane 2), HeLa (Lane 3), DU 145 (Lane 4), PC-3 (Lane 5) and SK-OV-3 (Lane 6). The blot was probed with Anti-ARF1/ARF3/ARF5/ARF6 Monoclonal Antibody (Product # MA3-060, 1:3000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/ml, 1:4000 dilution). A 18 kDa band corresponding to ARF1/ARF3/ARF5/ARF6 was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ARF1/ARF3/ARF5/ARF6 was achieved by transfecting HeLa with ARF1/ARF3/ARF5/ARF6 specific siRNAs (Silencer® select Product # s1555, s1552 ). Western blot analysis (Fig. a) was performed using whole cell extracts from the ARF1/ARF3/ARF5/ARF6 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ARF1/ARF3/ARF5/ARF6 Monoclonal Antibody (Product # MA3-060, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to ARF1/ARF3/ARF5/ARF6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ADP-Ribosylation Factor using ADP-Ribosylation Factor Monoclonal Antibody (1D9) (Product # MA3-060) shows staining in Hela Cells. ADP-Ribosylation Factor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing ADP-Ribosylation Factor (Product # MA3-060) at a dilution of 1:100 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ADP-Ribosylation Factor using ADP-Ribosylation Factor Monoclonal Antibody (1D9) (Product # MA3-060) shows staining in MCF-7 Cells. ADP-Ribosylation Factor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing ADP-Ribosylation Factor (Product # MA3-060) at a dilution of 1:100 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ADP-Ribosylation Factor using ADP-Ribosylation Factor Monoclonal Antibody (1D9) (Product # MA3-060) shows staining in U251 Cells. ADP-Ribosylation Factor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing ADP-Ribosylation Factor (Product # MA3-060) at a dilution of 1:100 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ARF1/ARF3/ARF5/ARF6 was performed using 70% confluent log phase MDA-MB-231 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ARF1/ARF3/ARF5/ARF6 Mouse Monoclonal Antibody (Product # MA3-060) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human colon carcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing ADP-Ribosylation Factor (Product # MA3-060) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human liver tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a mouse monoclonal antibody recognizing ADP-Ribosylation Factor (Product # MA3-060) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human tonsil tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a mouse monoclonal antibody recognizing ADP-Ribosylation Factor (Product # MA3-060) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of ADP-Ribosylation Factor in 3T3 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ADP-Ribosylation Factor monoclonal antibody (Product # MA3-060) at a dilution of 1:80 for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of ADP-Ribosylation Factor in Hela cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ADP-Ribosylation Factor monoclonal antibody (Product # MA3-060) at a dilution of 1:80 for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of ADP-Ribosylation Factor in MCF-7 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ADP-Ribosylation Factor monoclonal antibody (Product # MA3-060) at a dilution of 1:80 for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.