ARF1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 2 and 1 bp insertion in exon 2.
ADP-ribosylation factor 1, ARF1_HUMAN
ARF1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 2 and 1 bp insertion in exon 2.
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
ARF1 also known as ADP-ribosylation factor 1 or ARF1 protein is a small GTPase with a molecular mass of approximately 20 kDa. Commonly expressed in a variety of cell types ARF1 plays a central role in vesicle trafficking. As an activator ARF1 interacts with different GTPase-activating proteins such as ARFGAP1 and BIG1 ensuring the proper distribution of proteins and lipids in cells. ARF1's expression occurs across numerous cellular compartments including the Golgi apparatus aiding in the formation of transport vesicles.
ARF1 is essential in regulating membrane dynamics and vesicular traffic. It forms part of the COPI and clathrin-coated vesicle complexes where it recruits coat proteins to budding vesicles. This recruitment is fundamental for maintaining Golgi structure and function. Additionally ARF1 plays a role in cytokinesis by interacting with the septin cytoskeleton. The protein also influences actin cytoskeleton remodeling which is pivotal for cell shape changes and motility.
ARF1 is central to both the endocytic and secretory pathways. It collaborates with proteins like ARFS and 3F1 in modulating the trafficking of cargo between the endoplasmic reticulum and Golgi. Within the secretory pathway ARF1 interacts with SNARE proteins to facilitate vesicle docking and fusion. Its actions in pathways maintain cellular homeostasis and promote proper cellular response to various stimuli.
ARF1 has links to cancer and Alzheimer's disease. Overexpression of ARF1 correlates with tumor progression and metastasis impacting cell proliferation and survival mechanisms. Additionally its disruption associates with Alzheimer's where it may influence amyloid precursor protein processing alongside interactions with GAP proteins. Understanding ARF1's role in these conditions highlights its potential as a therapeutic target stressing the importance of studying its interactions with disease-related proteins.
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Anti-ARF1 antibody ab183576 was shown to react with ARF1 in wild-type HeLa cells in Western blot with loss of signal observed in ARF1 knockout cell line ab264939 (ARF1 knockout cell lysate Human ARF1 knockout HeLa cell lysate ab257353). Wild-type HeLa and ARF1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-ARF1 antibody ab183576 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-ARF1 antibody (Anti-ARF1 antibody ab183576) at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: ARF1 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human ARF1 knockout HeLa cell line (ab264939)
Lane 3: MDA-MB-231 cell lysate at 20 µg
Lane 4: PANC-1 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 18 kDa
Anti-ARF1 antibody ab58578 was shown to react with ARF1 in wild-type HeLa cells in Western blot with loss of signal observed in ARF1 knockout cell line ab264939 (ARF1 knockout cell lysate Human ARF1 knockout HeLa cell lysate ab257353). Wild-type HeLa and ARF1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-ARF1 antibody ab58578 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Donkey anti-Goat IgG H&L (IRDye® 800CW) preabsorbed (Donkey anti-Goat IgG H&L (IRDye® 800CW) preadsorbed ab216775) and Donkey anti-Mouse 680RD secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-ARF1 antibody (Anti-ARF1 antibody ab58578) at 1 µg/mL
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: ARF1 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human ARF1 knockout HeLa cell line (ab264939)
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 18 kDa
Allele-1: 1 bp deletion in exon 2.
Allele-2: 1 bp insertion in exon 2.
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