Human PDCD6IP knockout HEK-293 cell line (ab260864)
Overview
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Product name
Human PDCD6IP knockout HEK-293 cell line -
Parental Cell Line
HEK-293 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 1 bp insertion -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: ICC, WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HEK-293 cell line (ab259776). 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 (High Glucose) + 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 2x104 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 2x104 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.
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Kidney -
Cell type
epithelial -
Gender
Female -
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
Class E VPS protein involved in concentration and sorting of cargo proteins of the multivesicular body (MVB) for incorporation into intralumenal vesicles (ILVs) that are generated by invagination and scission from the limiting membrane of the endosome. Binds to the phospholipid lysobisphosphatidic acid (LBPA) which is abundant in MVBs internal membranes. The MVB pathway appears to require the sequential function of ESCRT-O, -I,-II and -III complexes. The ESCRT machinery also functions in topologically equivalent membrane fission events, such as the terminal stages of cytokinesis and enveloped virus budding (HIV-1 and other lentiviruses). Appears to be an adapter for a subset of ESCRT-III proteins, such as CHMP4, to function at distinct membranes. Required for completion of cytokinesis. Involved in HIV-1 virus budding. Can replace TSG101 it its role of supporting HIV-1 release; this function implies the interaction with CHMP4B. May play a role in the regulation of both apoptosis and cell proliferation. -
Sequence similarities
Contains 1 BRO1 domain. -
Cellular localization
Cytoplasm > cytosol. Melanosome. Cytoplasm > cytoskeleton > centrosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. Colocalized with CEP55 in the midbody during cytokinesis. Colocalized with CEP55 at centrosomes of non-dividing cells. - 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 ab260864 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 |
|---|---|---|
| ICC |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration.
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| Notes |
|---|
|
ICC
Use at an assay dependent concentration. |
|
WB
Use at an assay dependent concentration. |
Images
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Western blot - Human PDCD6IP knockout HEK-293 cell line (ab260864)All lanes : Anti-ALIX antibody (ab88388) at 1 µg/ml
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : ALIX knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab88388 observed at 96 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab88388 was shown to recognize in wild-type HEK-293 cells as signal was lost at the expected MW in ALIX knockout cell line ab260864 (knockout cell lysate ab261656). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and ALIX knockout samples were subjected to SDS-PAGE. ab88388 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.
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Immunocytochemistry - Human PDCD6IP knockout HEK-293 cell line (ab260864)ab88388 staining ALIX in wild-type HEK-293 cells (top panel) and ALIX knockout HEK-293 cells (ab260864) (bottom panel). The cells were fixed with 100% methanol (5 min), 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 ab88388 at 5µg/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 pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).
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Western blot - Human PDCD6IP knockout HEK-293 cell line (ab260864)All lanes : Anti-ALIX antibody [EPR15314-33] - N-terminal (ab186728) at 1/5000 dilution
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : ALIX knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab186728 observed at 96 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab186728 was shown to recognize in wild-type HEK-293 cells as signal was lost at the expected MW in ALIX knockout cell line ab260864 (knockout cell lysate ab261656). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and ALIX knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. ab186728 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab260864 has not yet been referenced specifically in any publications.