PA1-127

antibody from Invitrogen Antibodies

Targeting: ARF1

Provider product page for PA1-127

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [3]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [2]
Immunohistochemistry [3]
Other assay [2]

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Product number: PA1-127 - Provider product page
Provider: Invitrogen Antibodies
Product name: ARF1 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-127 has been successfully used in Western blot in human, mouse, and canine samples, and in immunofluorescence and immunoprecipitation applications in human samples.
Reactivity: Human, Mouse, Canine
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20°C

ARF1 protein structure - PA1-127 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Arf1 was performed by loading 25 µg of various whole cell lysates per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Arf1 polyclonal antibody (Product # PA1-127) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-rabbit IgG-HRP secondary antibody (Product # 31460) at a dilution of 1:15,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Arf1 was performed by loading 25 µg of various whole cell lysates per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Arf1 polyclonal antibody (Product # PA1-127) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-rabbit IgG-HRP secondary antibody (Product # 31460) at a dilution of 1:15,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

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), MDA-MB-231 treated with Brefeldin (5 µg/ml for 60 min) (Lane 2), HeLa (Lane 3), U-2 OS (Lane 4), DU 145 (Lane 5), PC-3 (Lane 6) and SK-OV-3 (Lane 7). The blot was probed with Anti-ARF1 Polyclonal Antibody (Product # PA1-127, 1:3000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 18 kDa band corresponding to ARF1 was observed across the cell lines tested and enhanced upon Brefeldin treatment in MDA-MB-231 cell line.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ARF1 was achieved by transfecting HeLa with ARF1 specific siRNAs (Silencer® select Product # s1552 ). Western blot analysis (Fig. a) was performed using whole cell extracts from the ARF1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ARF1 Polyclonal Antibody (Product # PA1-127, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Arf1 in HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 5% normal goat serum (Product # 31873) for 15 minutes at room temperature. Cells were probed with an Arf1 polyclonal antibody (Product # PA1-127) 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-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ARF1 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 Rabbit Polyclonal Antibody(Product # PA1-127) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 predominantly Golgi complex 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 analysis of Arf1 showing staining in the cytoplasm of paraffin-embedded human breast carcinoma (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 Arf1 polyclonal antibody (Product # PA1-127) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C 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 Arf1 showing staining in the cytoplasm of paraffin-embedded human colon carcinoma (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 Arf1 polyclonal antibody (Product # PA1-127) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C 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 Arf1 showing staining in the cytoplasm of paraffin-embedded mouse colon 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 Arf1 polyclonal antibody (Product # PA1-127) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C 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: Immunoprecipitation of Arf1 was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 500 µg of HeLa whole cell lysate with 2 µg of an Arf1 polyclonal antibody (Product # PA1-127) overnight on a rocking platform at 4øC. The immune complexes were captured on 50 µL Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Samples, including the HeLa cell lysate as a positive control (left lane), were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a nitrocellulose membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a Arf1 polyclonal antibody (Product # PA1-127) at a dilution of 1:1000 overnight rotating at 4øC, washed in TBST, and probed with Clean-blot IP detection reagent (Product # 21230) at a dilution of 1:2500 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 PCLX-001 treatment attenuates BCR downstream signaling events in BL2 lymphoma cells. Western blot of BL2 cells treated for 48 h with 0.1 uM or 1.0 muM of dasatinib, ibrutinib or PCLX-001 to detect total tyrosine phosphorylation (P-Tyr), Lyn, Lyn phosphorylated on tyrosine 396 or 507, BTK, and BTK phosphorylated on tyrosines 223 or 551 ( a ), HGAL, SYK, phosphorylated SYK (P-SYK) ( b ) or ERK, phosphorylated ERK (P-ERK), NFkappaB, c-Myc, CREB, Arf-1, BIP, and PARP-1 ( c ). Western blots are representative of at least three independent experiments. GAPDH serves as a loading control. BL2 cells were activated with 25 mug/mL F(ab') 2 anti-human IgM for 2 min and processed for western blotting. All western blots shown are representative of three independent experiments. Source data are provided as a Source Data file.