Product nameHuman CENPB knockout A-431 cell line
Parental Cell LineA431
Mutation descriptionKnockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift: 99%
Knockout validationNext Generation Sequencing (NGS), Western Blot (WB)
Tested applicationsSuitable for: WBmore details
Recommended control: Human wild-type A-431 cell line (ab263975). 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.
- 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.
Number of cells1 x 106 cells/vial, 1 mL
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
PurityImmunogen affinity purified
FunctionInteracts with centromeric heterochromatin in chromosomes and binds to a specific subset of alphoid satellite DNA, called the CENP-B box. May organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes.
Sequence similaritiesContains 1 HTH CENPB-type DNA-binding domain.
Contains 1 HTH psq-type DNA-binding domain.
Cellular localizationNucleus. Chromosome > centromere.
- Information by UniProt
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab274919 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use at an assay dependent concentration.
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift: 99%
All lanes : Anti-CENPB antibody (ab25734) at 1 µg/ml
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2 : CENPB knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg
Lane 4 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 77 kDa why is the actual band size different from the predicted?
ab25734 was shown to react with CENPB in wild-type A-431 cells in Western blot with loss of signal observed in CENPB knockout cell line ab274919 (knockout cell lysate ab274977).Wild-type A-431 and CENPB knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab25734 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml 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.
ab274919 has not yet been referenced specifically in any publications.