Human RAB8A knockout HeLa cell line
- Advanced Validation
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RAB8A KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 423 bp deletion in exon 1. To order both knockout and wild-type control cells: select 2 x 1000000Cells/vial. To order only knockout cells: select 1000000Cells/vial.
View Alternative Names
AA409338, MEL, MGC124948, Mel transforming oncogene, Mel transforming oncogene (RAB8 homolog), Mel transforming oncogene (derived from cell line NK14), Mel transforming oncogene (derived from cell line NK14) RAB8 homolog, Oncogene c-mel, RAB8, RAB8A member RAS oncogene family, RAB8A_HUMAN, Ras associated protein RAB8, Ras-related protein Rab-8A
- ICC/IF
Supplier Data
Immunocytochemistry/ Immunofluorescence - Human RAB8A knockout HeLa cell line (AB264993)
ab237702 was shown to react with RAB8A in wild-type HeLa cells in immunocytochemistry with loss of signal observed in RAB8A knockout cell line ab264993. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab237702 at 1/250 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 ug/ml.
Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
- ICC/IF
Supplier Data
Immunocytochemistry/ Immunofluorescence - Human RAB8A knockout HeLa cell line (AB264993)
ab241061 was shown to react with RAB8A in wild-type HeLa cells in immunocytochemistry with loss of signal observed in RAB8A knockout cell line ab264993. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab241061 at 1/600 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 ug/ml.
Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
- WB
Supplier Data
Western blot - Human RAB8A knockout HeLa cell line (AB264993)
ab188574 was shown to react with RAB8A in wild-type HeLa cells in Western blot with loss of signal observed in RAB8A knockout cell line ab264993. Wild-type HeLa and RAB8A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab188574 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
All lanes:
Western blot - Anti-RAB8A antibody [EPR14873] (<a href='/en-us/products/primary-antibodies/rab8a-antibody-epr14873-ab188574'>ab188574</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa lysate at 30 µg
Lane 2:
RAB8A knock-out HeLa lysate at 30 µg
Lane 2:
Western blot - Human RAB8A knockout HeLa cell line (ab264993)
false
- WB
Supplier Data
Western blot - Human RAB8A knockout HeLa cell line (AB264993)
ab241061 was shown to react with RAB8A in wild-type HeLa cells in Western blot with loss of signal observed in RAB8A knockout cell line ab264993. Wild-type HeLa and RAB8A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab241061 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
All lanes:
Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (<a href='/en-us/products/primary-antibodies/rab8a-antibody-mjf-r22-79-3-ab241061'>ab241061</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa lysate at 30 µg
Lane 2:
RAB8A knock-out HeLa lysate at 30 µg
Lane 2:
Western blot - Human RAB8A knockout HeLa cell line (ab264993)
false
- WB
Supplier Data
Western blot - Human RAB8A knockout HeLa cell line (AB264993)
ab237702 was shown to react with RAB8A in wild-type HeLa cells in Western blot with loss of signal observed in RAB8A knockout cell line ab264993. Wild-type HeLa and RAB8A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab237702 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
All lanes:
Western blot - Anti-RAB8A antibody [MJF-R22] (<a href='/en-us/products/primary-antibodies/rab8a-antibody-mjf-r22-ab237702'>ab237702</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa lysate at 30 µg
Lane 2:
RAB8A knock-out HeLa lysate at 30 µg
Lane 2:
Western blot - Human RAB8A knockout HeLa cell line (ab264993)
false
- WB
Lab
Western blot - Human RAB8A knockout HeLa cell line (AB264993)
Lanes 1-4 : Merged signal (red and green). Green - ab188574 observed at 24 kDa. Red - loading control ab8245 observed at 37 kDa.
ab188574 Anti-Rab8A antibody [EPR14873] was shown to specifically react with Rab8A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264993 (knockout cell lysate ab257195) was used. Wild-type and Rab8A knockout samples were subjected to SDS-PAGE. ab188574 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-RAB8A antibody [EPR14873] (<a href='/en-us/products/primary-antibodies/rab8a-antibody-epr14873-ab188574'>ab188574</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
RAB8A knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human RAB8A knockout HeLa cell line (ab264993)
Lane 3:
HCT116 cell lysate at 20 µg
Lane 4:
Human brain tissue lysate at 20 µg
Predicted band size: 24 kDa
Observed band size: 24 kDa
false
- WB
Supplier Data
Western blot - Human RAB8A knockout HeLa cell line (AB264993)
Lanes 1-4 : Merged signal (red and green). Green - ab241061 observed at 24 kDa. Red - loading control ab8245 observed at 37 kDa.
ab241061 Anti-Rab8A antibody [MJF-R22-79-3] was shown to specifically react with Rab8A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264993 (knockout cell lysate ab257195) was used. Wild-type and Rab8A knockout samples were subjected to SDS-PAGE. ab ab241061 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (<a href='/en-us/products/primary-antibodies/rab8a-antibody-mjf-r22-79-3-ab241061'>ab241061</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
RAB8A knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human RAB8A knockout HeLa cell line (ab264993)
Lane 3:
HCT116 cell lysate at 20 µg
Lane 4:
Human brain tissue lysate at 20 µg
Predicted band size: 24 kDa
Observed band size: 24 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human RAB8A knockout HeLa cell line (AB264993)
Homozygous : 423 bp deletion in exon 1.
- Cell Culture
Unknown
Cell Culture - Human RAB8A knockout HeLa cell line (AB264993)
Representative images of RAB8A knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.
- ICC/IF
Supplier Data
Immunocytochemistry/ Immunofluorescence - Human RAB8A knockout HeLa cell line (AB264993)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAB8A KO HeLa (RAB8A knockout human cervical adenocarcinoma epithelial cell) (ab264993) cells labeling RAB8A with ab307607 at 1/500 dilution, followed by ab98712 Goat Anti-Mouse IgG Fc (DyLight® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing cytoplasmic staining in Parental HeLa cell line, and no staining in RAB8A KO HeLa cell line. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/50 dilution (10 µg/ml) (Red). Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab98712 Goat Anti-Mouse IgG Fc (DyLight® 488) preadsorbed at 1/1000 dilution (2 µg/ml).
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
Initial handling guidelines
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.
Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
The small GTPase RAB8A contributes to the biosynthetic transport from the trans-Golgi network to the plasma membrane. It performs this activity in coordination with other proteins as part of a larger complex. RAB8A also plays an essential role in cilia formation and maintenance affecting cell polarity and signaling functions. The protein's ability to regulate vesicle movement makes it important for cellular homeostasis and communication.
Pathways
The regulatory function of RAB8A is significant in the ciliary membrane trafficking pathway and the insulin signaling pathway. Within these pathways RAB8A interacts closely with other proteins like Rab11 and Rabin8 which help support its roles in directing vesicle transport. These interactions are essential for proper cellular signaling and the maintenance of polarized cellular structures further supporting the function of various cellular pathways.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
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Advanced Validation
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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