Human ANXA6 (Annexin-6) knockout HeLa cell line (ab265677)
Overview
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Product name
Human ANXA6 (Annexin-6) knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 7 and 5 bp deletion in exon 7 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab255928). 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
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10 -
Antibiotic resistance
Puromycin 1.00µg/ml -
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 associate with CD21. May regulate the release of Ca(2+) from intracellular stores. -
Sequence similarities
Belongs to the annexin family.
Contains 8 annexin repeats. -
Domain
A pair of annexin repeats may form one binding site for calcium and phospholipid. -
Post-translational
modificationsPhosphorylated in response to growth factor stimulation. -
Cellular localization
Cytoplasm. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
- Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422)
- Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023)
- Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024)
- Anti-Annexin-6/ANXA6 antibody [EPR19517] - BSA and Azide free (ab240359)
- Anti-Annexin-6/ANXA6 antibody [EPR19536] - BSA and Azide free (ab251329)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab265677 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: 76 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 76 kDa. |
Images
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All lanes : Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 76 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab199422 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab199422 was shown to react with Annexin-6/ANXA6 in wild-type HeLa cells in western blot with loss of signal observed in ANXA6 knockout cell line ab265677 (ANXA6 knockout cell lysate ab257351). Wild-type and ANXA6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab199422 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 hour at room temperature before imaging.
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All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 76 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab201023 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.
ab201023 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with 6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used. Wild-type and 6/ANXA6 knockout samples were subjected to SDS-PAGE. ab201023 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 76 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab201024 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.
ab201024 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with Annexin-6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used. Wild-type and Annexin-6/ANXA6 knockout samples were subjected to SDS-PAGE. ab201024 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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 20000 dilution for 1 hour at room temperature before imaging.
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Allele-1: 5 bp deletion in exon 7.
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Allele-2: 1 bp insertion in exon 7.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab265677 has not yet been referenced specifically in any publications.