Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)
Overview
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Product name
Human TGOLN2 (TGN46) knockout A549 cell line -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp deletion, 1 bp insertion; Frameshift: 100% -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, ICCmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A549 cell line (ab259777). 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: DMEM:Hams F12 + 5% 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, 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.
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
May be involved in regulating membrane traffic to and from trans-Golgi network. -
Tissue specificity
Isoform TGN46 is widely expressed. Isoform TGN51 is more abundant in fetal lung and kidney. Isoform TGN48 is barely expressed in embryonic kidney and promyelocytic cells. -
Cellular localization
Cell membrane. Golgi apparatus > trans-Golgi network membrane. Primarily in trans-Golgi network. Cycles between the trans-Golgi network and the cell surface returning via endosomes. - 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 ab269510 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 51 kDa.
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| ICC |
Use at an assay dependent concentration.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 51 kDa. |
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ICC
Use at an assay dependent concentration. |
Images
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Western blot - Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)All lanes : Anti-TGN46 antibody [EPR12676] (ab174280) at 1/1000 dilution
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : TGOLN2 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 51 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab174280 observed at 80 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab174280 was shown to react with TGN46 in wild-type A549 cells in Western blot with loss of signal observed in TGOLN2 knockout cell line ab269510 (knockout cell lysate ab269672). Wild-type A549 and TGOLN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab174280 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
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Next Generation Sequencing - Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)Knockout achieved by CRISPR/Cas9; X = 1 bp deletion, 1 bp insertion; Frameshift: 100%
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Western blot - Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)All lanes : Anti-TGN46 antibody [EPR24770-16] (ab271183) at 1/1000 dilution
Lane 1 : Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg
Lane 2 : TGOLN2 (TGN46) knockout A549 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg
Lane 3 : HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at a 1/10000 dilution.
Predicted band size: 51 kDa
Observed band size: 80-100 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 3% NFDM/TBST.
Lanes 1-3: Merged signal (red and green).
Green: ab271183 observed at 80-100 kDa.
Red: loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
Lanes 1-2: ab271183 was shown to react with TGN46 in wild-type A549 cells in Western blot with loss of signal observed in TGN46 knockout cell line ab269510 (knockout cell lysate ab269672). Wild-type A549 and TGN46 knockout cell lysates were subjected to SDS-PAGE.
ab271183 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1/1000 dilution and 1/20000 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 1/10000 dilution for 1 hour at room temperature before imaging.
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Immunocytochemistry - Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)ab41702 staining TGN46 in wild-type A549 cells (top panel) and TGOLN2 knockout A549 cells (bottom panel) (ab269510). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab41702 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry - Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)ab174280 staining TGN46 in wild-type A549 cells (top panel) and TGOLN2 knockout A549 cells (bottom panel) (ab269510). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab174280 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry - Human TGOLN2 (TGN46) knockout A549 cell line (ab269510)ab50595 staining TGN46 in wild-type A549 cells (top panel) and TGOLN2 knockout A549 cells (bottom panel) (ab269510). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab50595 at 0.1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab269510 has not yet been referenced specifically in any publications.