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ARF6 protein, human

The ARF6 protein is a small GTPase that plays a key role in regulating membrane trafficking and cytoskeleton organization within cells.
It is involved in a variety of cellular processes, such as endocytosis, cell migration, and cytokinesis.
Unraveling the mysteries surrounding the ARF6 protein can provide valuable insights into its function and potential therapeutic applications.
PubCompare.ai's AI-powered platform can help optimize your research protocols, identify the most reliable and effective procedures from literature, preprints, and patents, and streamline your research for enhanced reproducibility and accuracy.
Elevate your findings and discover new insights into the ARF6 protien with PubCompare.ai.

Most cited protocols related to «ARF6 protein, human»

1
Quantifying Exosomal Proteins by Western Blot
Cited 14 times
Protein-based quantitation of isolated exosomes was done using the protein DC assay kit as described earlier by us7 (link),34 (link). Subsequently, equal amount of exosomes (25 μg) collected from different isolation methods was denatured using 6X denaturation buffer at 95 °C for 10 min and then resolved on 12% SDS-Polyacrylamide gel by electrophoresis. Resolved proteins were transferred onto Immobilon-P PVDF membrane and then blocked by incubating in 5% skimmed milk to minimize non-specific binding of antibodies. Blocked blots were submerged with primary antibodies for CD9 and ARF-6 overnight, and subsequently washed three times (10 min each) with 1X Tris buffer saline with 0.1% Tween-20 (TBST) buffer followed by incubation with HRP-conjugated secondary anti-mouse (for CD9) and anti-rabbit (for ARF-6) antibodies. Unbound antibodies were removed by washing with 1X TBST buffer (3 × 10 min), and signal recorded using WestFemto maximum sensitivity substrate kit under Bio-Rad ChemiDoc Imager (Hercules, CA, USA).
Patel G.K., Khan M.A., Zubair H., Srivastava S.K., Khushman M., Singh S, & Singh A.P. (2019). Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Scientific Reports, 9, 5335.
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Publication 2019
Antibodies ARF6 protein, human Biological Assay Buffers Electrophoresis Exosomes Hypersensitivity Immobilon P isolation Mice, House Milk, Cow's polyacrylamide gels polyvinylidene fluoride Proteins Rabbits Saline Solution Tissue, Membrane Tromethamine Tween 20
2
Purification and Labeling of Proteins
Cited 13 times
GST fusion and 6x-his-tagged recombinant proteins were purified as previously described 19 (link). AP-2 and clathrin were purified as previously described 52 (link). Alexa 546-conjugated Tf, Alexa 555-EGF, and Dil-LDL, and Alexa555-conjugated forms of dextran and CTB were obtained (Invitrogen).
The following antibodies were used, with dilution and source as indicated. Antibodies against the following proteins have been described previously 28 (link), 53 (link): ARF6 (1:200), ARFGAP1 (1:1000), BARS (1:2000), β2-adaptin (AP.6, 1:5000), β-COP (M3A5, 1:100, or culture supernatant, 1:10), CHC (TD.1, 1:500, or culture supernatant, 1:30), giantin (1:5000), Lamp1 (1:500), Myc epitope (9E10, 1:1000), TfR (5E9, 1:500, or H68.4, 1:1000), and VSVG (BW8G65, culture supernatant 1:10). Antibodies against other proteins were obtained: CD8 (1:400, Santa Cruz), EEA1 (1:200, BD Bioscience), HA epitope (HA.11, 1:1500, Covance), and TGN46 (1:1000, AbD Serotec).
Bai M., Gad H., Turacchio G., Cocucci E., Yang J.S., Li J., Beznoussenko G.V., Nie Z., Luo R., Fu L., Collawn J.F., Kirchhausen T., Luini A, & Hsu V.W. (2011). ARFGAP1 promotes endocytosis regulated by AP-2. Nature cell biology, 13(5), 559-567.
Publication 2011
ADRB2 protein, human Antibodies ARF6 protein, human Clathrin Dextran Epitopes lysosomal-associated membrane protein 1, human macrogolgin Proteins Recombinant Proteins Technique, Dilution
3
Measuring Endogenous ARF and Rac Activation
Cited 12 times
Activation levels of endogenous Rac were assayed in cells infected with ARNO-expressing adenoviruses as described above. Endogenous GTP-Rac was isolated by incubation with a GST fusion to the PBD domain of PAK and quantitated as described previously (Criss et al., 2001 (link)).
Activation of endogenous ARFs by ARNO expression was assayed using a novel pulldown assay. Cells were infected with ARNO-expressing adenoviruses in serum-free DME in the presence or absence of doxycycline as described above for 2 h. 20 ng/ml doxycycline was then added and the cells were incubated for an additional hour. Extensive ruffling and migration of the ARNO-expressing cells was apparent at this point. Cells were then lysed at 4°C in 0.65 ml of 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol with 0.1 mM PMSF and 1 μg/ml each pepstatin, leupeptin, and antipain. Lysates were clarified by centrifugation at 16 g for 2 min in the presence of CL-4B Sepharose beads (Amersham Pharmacia Biotech). 0.5 ml of the clarified lysate was incubated with 40 μg of GST-GGA3 bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 30 min. The beads were then washed three times with 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1% NP-40, 10% glycerol with 0.1 mM PMSF, and 1 μg/ml each of pepstatin, leupeptin, and antipain. Bound proteins were eluted into 60 μl SDS-PAGE sample buffer. This sample was divided (15 μl for ARF1 and 40 μl for ARF6) and assayed for the presence of endogenous ARF1 and ARF6 by Western blotting with ARF1- and ARF6-specific antibodies. Total levels of each ARF in the starting lysates were assayed by Western blotting of 3% of the clarified lysate.
Santy L.C, & Casanova J.E. (2001). Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. The Journal of Cell Biology, 154(3), 599-610.
Publication 2001
Adenoviruses Antibodies Antipain ARF6 protein, human Buffers Cells Centrifugation Deoxycholic Acid, Monosodium Salt Doxycycline Glutathione Glycerin leupeptin Magnesium Chloride Migration, Cell Nonidet P-40 pepstatin Proteins SDS-PAGE Sepharose Sepharose CL 4B Serum Sodium Chloride Triton X-100 Tromethamine
4
Recombinant Protein Preparation and Kinase Assay
Cited 8 times
Recombinant protein preparation and cloning were done using standard methods. In immunofluorescence experiments, we transfected cells with Fugene 200 or microinjected them with 10 nM protein, where indicated. We performed kinase assays with rabbit pEGFP–PAK2, which was immunoprecipitated from 293T cell lysates and incubated with the protein of interest in the presence of MBP, 10 µM ATP and 5 µCi γ32P[ATP]. Reactions were stopped by the addition of SDS buffer, separated by SDS–polyacrylamide gel electrophoresis and kinase activity was measured as 32P counts per minute. EspG–ARF6 and EspG–PAK2–Iα3 were purified by ion exchange and gel filtration chromatography and crystallized by the hanging-drop vapour diffusion method. We collected X-ray diffraction data at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory (USA). The structure of EspG–ARF6 was phased to a resolution of 2.5 Å by the multiwavelength anomalous dispersion method using selenomethionine-labelled EspG and ARF6 proteins. The EspG–PAK2 structure was solved to a resolution of 2.8 Å by the molecular replacement method using the EspG monomer of the EspG–ARF6 structure as the initial search model. Further details can be found in Supplementary Information.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.
Selyunin A.S., Sutton S.E., Weigele B.A., Reddick L.E., Orchard R.C., Bresson S.M., Tomchick D.R, & Alto N.M. (2010). The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold. Nature, 469(7328), 107-111.
Publication 2010
ADP-Ribosylation Factor 6 ARF6 protein, human Biological Assay Buffers Cells Diffusion FuGene Gel Chromatography HEK293 Cells Immunofluorescence Ion Exchange PAK2 Kinase Phosphotransferases Proteins Rabbits Recombinant Proteins SDS-PAGE Selenomethionine X-Ray Diffraction
5
Antibody Profiling for Cell Biology
Cited 6 times

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Publication 2009
alpha-Tubulin Antibodies Antibodies, Anti-Idiotypic ARF6 protein, human BDP1 protein, human Caspase 3 CD29 Antigen CTTN protein, human Genes, MHC Class I Hybridomas Immunoglobulins Plasmids Vesicle-Associated Membrane Protein 3

Most recents protocols related to «ARF6 protein, human»

1
GGA3-mediated Pulldown of ARF Proteins
Method described previously (Nacke et al., 2021 (link)); briefly, PC3 cells were incubated on 120-mm plates for 48 h at 37°C, 5% CO2. Cells were then lysed on ice in pulldown-lysis buffer (50 mM Tris, 100 mM NaCl, 2 mM MgCl2, 0.1% SDS, 0.5% Na-deoxycholate, 1% Triton X-100, 10% glycerol). Lysates were syringed 5× using a 25-27G needle and centrifuged at 4°C 14,000 g for 1 min. Spin columns were equilibrated with 50 μl of Glutathione Agarose resin and washed with pulldown-column wash buffer (1:1 pulldown-lysis buffer and 1×TBS). 80 μg of GST-GGA3-GAT recombinant fusion protein was immobilized on the agarose resin by incubation at 4°C with gentle rocking. After 1 h, 1 mg of each lysate was added onto spin columns and incubated again at 4°C for 2 h with rocking. Pulldown wash buffer (50 mM Tris, 100 mM NaCl, 2 mM MgCl2, 1% NP-40, 10% glycerol) was used to wash unbound proteins off the column. 60 μl of pulldown-elution buffer (10 mM Glutathione in 1×TBS) was added to each spin column and incubated for 5 min at room temperature. Eluted protein was collected at 1,250 g for 1 min and samples prepared for SDS-PAGE and immunoblotting as described above. n = 3 experimental replicates for ARF3 Chimeras and n = 5 for PSD KD. Data is presented as GGA3 binding normalized to ARF3 or ARF6 levels for each experiment. Values are mean ± SEM.
Sandilands E., Freckmann E.C., Cumming E.M., Román-Fernández A., McGarry L., Anand J., Galbraith L., Mason S., Patel R., Nixon C., Cartwright J., Leung H.Y., Blyth K, & Bryant D.M. (2023). The small GTPase ARF3 controls invasion modality and metastasis by regulating N-cadherin levels. The Journal of Cell Biology, 222(4), e202206115.
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Publication 2023
ARF6 protein, human Buffers Cells Chimera Deoxycholate Glutathione Glycerin Magnesium Chloride Needles Nonidet P-40 PC 3 Cell Line Proteins Recombinant Fusion Proteins Resins, Plant SDS-PAGE Sepharose Sodium Chloride Triton X-100 Tromethamine
2
HUVEC Lysis and Western Blotting
HUVEC cultures were trypsinized and lysed using Ripa buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin) containing 1× ProBlock™ Protease Inhibitor Cocktail-50 (GoldBio) and processed as previously described[17 (link)]. Protein was then transferred to Immun-Blot PVDF Membrane at 4°C, 100 V for 1 hour 10 minutes. Blots were blocked in 2% milk proteins for 1 hour, then put in primary antibody at specified concentrations overnight. After 3 10-minute washes with PBS, secondary antibodies at specified concentrations were applied for 4 hours. After 3 additional PBS washes, blots were developed with ECL reagent. Arf6 activation assay blots were performed using commercially available kits listed in the Supplemental Information.
Francis C.R., Bell M.L., Skripnichuk M.M, & Kushner E.J. (2023). Arf6 Regulates Endocytosis and Angiogenesis by Promoting Filamentous Actin Assembly. bioRxiv.
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Publication Preprint 2023
Antibodies ARF6 protein, human beta-glycerol phosphate Biological Assay Buffers Deoxycholic Acid, Monosodium Salt Edetic Acid Egtazic Acid Immunoglobulins leupeptin Milk Proteins Nonidet P-40 polyvinylidene fluoride Protease Inhibitors Proteins Radioimmunoprecipitation Assay Sodium Chloride sodium pyrophosphate Tissue, Membrane Tromethamine
3
Measuring Arf6-GTP in IQSEC2 Mutants
We measured Arf6-GTP in HEK293T cells transiently transfected with IQSEC2 carrying IQ domain mutations (A350V, A350T, A350D, R359C, or E849K IQSEC2) cloned into pCAGGS and HA-tagged Arf6 in pXS [10 (link)]. The relative amount of ARF6 bound to GTP as compared to total ARF6 was assessed using a GGA-3 pulldown assay and Western blot as previously described [46 (link)]. Bands were visualized using a LiCor Odyssey imaging system and quantified with Image Studio Lite. Each band was normalized to the untreated sham-transfected control.
Jada R., Borisov V., Laury E., Halpert S., Levy N.S., Wagner S., Netser S., Walikonis R., Carmi I., Berlin S, & Levy A.P. (2023). Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction. International Journal of Molecular Sciences, 24(4), 3924.
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Publication 2023
ARF6 protein, human Biological Assay Cells Mutation Western Blotting
4
Extracellular Vesicle Protein Analysis
The following antibodies were used in this study: anti-CD9 mouse monoclonal primary antibodies (clone 4A2, cat. SAB1402143) and anti-CD63 mouse monoclonal primary antibodies (clone RFAC4, cat. CBL553) from Sigma-Aldrich (St. Louis, MO, USA), as well as Hsp70 (clone C92F3A-5, cat. sc-66048) and mouse monoclonal primary antibodies for Arf6 (clone 3A-1, cat. sc-7971) from Santa Cruz Biotechnology (Dallas, TX, USA). Other reagents were obtained as listed in [17 (link)].
Surman M., Jankowska U., Wilczak M, & Przybyło M. (2023). Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry. Cancers, 15(4), 1097.
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Publication 2023
Anti-Antibodies Antibodies ARF6 protein, human Clone Cells Heat-Shock Proteins 70 Monoclonal Antibodies Mus
5
Exosome Isolation and Characterization
Before exosome isolation, CM cells were kept for 24 h in FBS-free media. Conditioned media were then collected (approx. 200 mL per exosome sample) and centrifuged. Through centrifugations at 400× g (5 min, 4 °C) and 4000× g (20 min, 4 °C), cells and cellular debris were removed; supernatants were then concentrated by a low-vacuum filtration (LVF) procedure described by Drożdż et al. [21 (link)]. Concentrated media (approx. 2 mL) were centrifuged three times: at 7000× g (20 min, 4 °C), 18,000× g (20 min, 4 °C), and 80,000× g (20 min, 4 °C), to remove any larger vesicles. The final centrifugation step was performed at 150,000× g (90 min, 4 °C) to obtain exosome pellets. Finally, exosomes were resuspended in ice-cold PBS or in a LC-MS/MS lysis buffer.
The purity of exosome samples was analyzed by nanoparticle tracking analysis (NTA) on NanoSight LM 10 (Malvern Panalytical, Malvern, UK). Five independent records were collected for each sample (10 μL diluted to 2 mL with PBS). The mean results ± SD are presented on graphs.
In addition, immunodetection of EV markers was performed. SDS-PAGE electrophoresis and WB (western blot) for each CM cell line and exosome sample were performed as described in [22 (link)]. The chosen markers were detected using anti-CD63 (1:2000), anti-CD9 (1:2000), anti-Hsp70 (1:2000), and anti-Arf6 (1:500) primary antibodies, and anti-mouse IgG-HRP (1:400) as a secondary antibody. Chemiluminescence-based detection was done using HRP substrates and ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).
Surman M., Jankowska U., Wilczak M, & Przybyło M. (2023). Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry. Cancers, 15(4), 1097.
Full text: Click here
Publication 2023
anti-IgG Antibodies ARF6 protein, human Buffers Cell Lines Cells Centrifugation Chemiluminescence Cold Temperature Culture Media, Conditioned Electrophoresis Exosomes Filtration Heat-Shock Proteins 70 Immunoglobulins isolation Mus Pellets, Drug SDS-PAGE Tandem Mass Spectrometry Vacuum Western Blotting

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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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Ab77581 by Abcam
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.
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The ChemiDoc Imaging System is a versatile lab equipment designed for the visualization and analysis of various biomolecules, such as proteins, nucleic acids, and chemiluminescent samples. It utilizes advanced imaging technologies to capture high-quality digital images for downstream applications.
8
Anti arf6 by Cell Signaling Technology
Sourced in China, United Kingdom
Anti-ARF6 is a laboratory reagent designed to detect and study the ARF6 protein. ARF6 is a member of the ADP-ribosylation factor (ARF) family of small GTPases, which play important roles in intracellular trafficking and cytoskeleton regulation. This antibody can be used to identify and quantify ARF6 in various experimental systems.
9
Anti arf6 by Abcam
Anti-ARF6 is a primary antibody that specifically recognizes the ARF6 protein. ARF6 is a small GTPase that plays a role in regulating membrane trafficking and cytoskeletal organization.
10
Arf6 activation assay kit by Cell Biolabs
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The Arf6 activation assay kit is a tool designed to measure the activation state of the ADP-ribosylation factor 6 (Arf6) protein. Arf6 is a small GTPase that plays a key role in various cellular processes, including membrane trafficking and cytoskeleton organization. The kit provides the necessary reagents and protocols to quantify the levels of active, GTP-bound Arf6 in cell lysates or tissue samples.

More about "ARF6 protein, human"

Unraveling the Mysteries of the ARF6 Protein: A Crucial Regulator of Cellular Processes.
The ARF6 (ADP-Ribosylation Factor 6) protein is a small GTPase that plays a pivotal role in governing membrane trafficking and cytoskeleton organization within cells.
This versatile protein is involved in a myriad of cellular processes, including endocytosis, cell migration, and cytokinesis.
Understanding the intricate functions of ARF6 can provide invaluable insights, leading to potential therapeutic applications.
PubCompare.ai's AI-powered platform can help optimize your research protocols, identifying the most reliable and effective procedures from the wealth of literature, preprints, and patents.
This streamlined approach can enhance the reproducibility and accuracy of your findings, enabling you to discover new insights into the ARF6 protein.
Leverage the power of cutting-edge technologies like Lipofectamine 2000, Ab77581, TRIzol reagent, FBS, β-actin, Bovine serum albumin, and the ChemiDoc Imaging System to uncover the secrets of this dynamic protein.
With the aid of PubCompare.ai's AI-driven comparisons, you can locate the most effective anti-ARF6 antibodies and Arf6 activation assay kits, empowering your research and elevating your discoveries.
Embark on a journey of scientific exploration and unlock the mysteries of the ARF6 protein with the support of PubCompare.ai.