Human RAB29 knockout A549 cell line (ab280040)
Overview
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Product name
Human RAB29 knockout A549 cell line -
Parental Cell Line
A549 -
Organism
Human -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A549 cell line (ab275463). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 10% FBS
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. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x103-1x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. 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 6x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not exceed 7x104 cells/cm2.
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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Rab GTPase key regulator in vesicle trafficking. Essential for maintaining the integrity of the endosome-trans-Golgi network structure. Together with LRRK2, plays a role in the retrograde trafficking pathway for recycling proteins, such as mannose 6 phosphate receptor (M6PR), between lysosomes and the Golgi apparatus in a retromer-dependent manner. Regulates neuronal process morphology in the intact central nervous system (CNS). May play a role in the formation of typhoid toxin transport intermediates during Salmonella enterica serovar Typhi (S.Typhi) epithelial cell infection. -
Tissue specificity
Ubiquitous. -
Sequence similarities
Belongs to the small GTPase superfamily. Rab family. -
Post-translational
modificationsIn case of Salmonella enterica serovar Typhimurium (S.Typhimurium) infection, is proteolytically cleaved between Gly-41 and Val-42 by the GtgE viral protease encoded on the Gifsy-2 lysogen bacteriophage, which therefore prevents the recruitment of RAB29 to S.Typhimurium-containing vacuoles. In contrast, no proteolytically cleavage is detected in S.Typhi-infected cells (PubMed:22042847). -
Cellular localization
Cell membrane. Cytoplasm. Cytoplasm, perinuclear region. Golgi apparatus. Golgi apparatus, trans-Golgi network. Vacuole. Cytoplasm, cytoskeleton. Colocalizes with LRRK2 along tubular structures emerging from Golgi apparatus (By similarity). Colocalizes with GM130 at the Golgi apparatus. Colocalizes with dynamic tubules emerging from and retracting to the Golgi apparatus. Colocalizes with TGN46 at the trans-Golgi network (TGN). In Salmonella enterica serovar Typhi (S.Typhi) infected epithelial cells, is recruited and colocalized with both S.Typhi-containing vacuoles and dynamic tubules as well as those emerging from the vacuole toward the cell periphery. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab280040 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 23 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 23 kDa. |
Images
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All lanes : Anti-RAB29 antibody [MJF-R30-104] (ab256527) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : RAB29 knockout A549 cell lysate
Lane 3 : MCF-7 cell lysate
Lane 4 : Caco-2 cell lysate
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDaFalse colour image of Western blot: Anti-RAB29 antibody [MJF-R30-104] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab256527 was shown to bind specifically to RAB29. A band was observed at 23 kDa in wild-type A549 cell lysates with no signal observed at this size in RAB29 knockout cell line ab280040 (knockout cell lysate ab280099). To generate this image, wild-type and RAB29 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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All lanes : Anti-RAB29 antibody [MJF-R30-124] (ab256526) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : RAB29 knockout A549 cell lysate
Lane 3 : MCF-7 cell lysate
Lane 4 : Caco-2 cell lysate
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDaFalse colour image of Western blot: Anti-RAB29 antibody [MJF-R30-124] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab256526 was shown to bind specifically to RAB29. A band was observed at 23 kDa in wild-type A549 cell lysates with no signal observed at this size in RAB29 knockout cell line ab280040 (knockout cell lysate ab280099). To generate this image, wild-type and RAB29 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Human RAB29 KO in A549 Cells with 119 bp Deletion in Exon 4
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab280040 has not yet been referenced specifically in any publications.