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AB265277

Human BAK1 (Bak) knockout HeLa cell line

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BAK1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 2 and 2 bp deletion in exon 2 and Insertion of the selection cassette in exon 2. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
8 Images
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Immunohistochemical analysis of paraffin-embedded fixed (A) Wild-type HeLa (human cervix adenocarcinoma epithelial cell) cell pellet. (B) BAK1 knockout HeLa (ab265277) cell pellet staining Bak with ab32371 at 1/2000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Counter-staining used was hematoxylin. Positive staining on (A) Wild-type HeLa cell pellet, no staining on (B) BAK1 knockout HeLa (ab265277) cell pellet. The section was incubated with ab32371 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Western blot - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • WB

Lab

Western blot - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Lanes 1- 2 : Merged signal (red and green). Green - ab32371 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32371 was shown to react with Bak in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265277 (knockout cell lysate ab257077) was used. Wild-type HeLa and BAK1knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32371 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

All lanes:

Western blot - Anti-Bak antibody [Y164] (<a href='/en-us/products/primary-antibodies/bak-antibody-y164-ab32371'>ab32371</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BAK1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human BAK1 (Bak) knockout HeLa cell line (ab265277)

Predicted band size: 23 kDa

Observed band size: 23 kDa

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Western blot - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • WB

Lab

Western blot - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Lanes 1- 2 : Merged signal (red and green). Green - ab92999 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab92999 was shown to react with Bak in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265277 (knockout cell lysate ab257077) was used. Wild-type HeLa and BAK1knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab92999 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at 1 µg/ml and a 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.

All lanes:

Western blot - Anti-Bak antibody (<a href='/en-us/products/primary-antibodies/bak-antibody-ab92999'>ab92999</a>) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BAK1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human BAK1 (Bak) knockout HeLa cell line (ab265277)

Predicted band size: 23 kDa

Observed band size: 23 kDa

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Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • Sanger seq

Unknown

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Allele-1 : 2 bp deletion in exon 2.

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • Sanger seq

Unknown

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Allele-3 : Insertion of the selection cassette in exon 2.

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • Sanger seq

Lab

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Sequencing chromatogram displaying sequence edit in exon 2

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • Sanger seq

Unknown

Sanger Sequencing - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Allele-2 : 1 bp deletion in exon 2.

Cell Culture - Human BAK1 (Bak) knockout HeLa cell line (AB265277)
  • Cell Culture

Unknown

Cell Culture - Human BAK1 (Bak) knockout HeLa cell line (AB265277)

Representative images of BAK1 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.

Key facts

Cell type

HeLa

Species or organism

Human

Tissue

Cervix

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 2 and 2 bp deletion in exon 2 and Insertion of the selection cassette in exon 2

Disease

Adenocarcinoma

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Complementary Products

Primary Antibodies

AB32371

Anti-Bak antibody [Y164]

BOND RX™ Validated|20ul selling size|RabMAb|Recombinant|KO Validated

Immunocytochemistry/ Immunofluorescence - Anti-Bak antibody [Y164] (AB32371)

  • 5
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Proteins & Peptides

AB95933

Recombinant human XIAP protein

Functional Studies - Recombinant human XIAP protein (AB95933)

  • 0
  • 0
Primary Antibodies

AB32503

Anti-Bax antibody [E63]

BOND RX™ Validated|20ul selling size|RabMAb|Recombinant|KO Validated

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bax antibody [E63] (AB32503)

  • 4
  • 12
Primary Antibodies

AB32158

Anti-Bim antibody [Y36]

RabMAb|Recombinant|KO Validated|20ul selling size

Flow Cytometry (Intracellular) - Anti-Bim antibody [Y36] (AB32158)

  • 4
  • 6
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Reactivity data

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Product details

We will provide viable cells that proliferate on revival.

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.

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What's included?

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Properties and storage information

Gene name
BAK1
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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Handling procedures

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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
  • Cells should be passaged when they have achieved 80-90% confluence.
Culture medium

DMEM (High Glucose) + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

The Bak protein also known as Bcl-2 homologous antagonist killer plays a mechanical role in the mitochondrial apoptosis pathway. It often functions as a pro-apoptotic member of the Bcl-2 protein family. Bak is expressed widely in the body being present in tissues such as the brain and heart. The molecular weight of Bak is approximately 23 kilodaltons. Bak interacts with other mitochondrial proteins to trigger cell death processes.
Biological function summary

Bak engages in promoting apoptosis by disrupting the mitochondrial outer membrane potential. It is part of the Bcl-2 family complex which balances cell survival and cell death. Bak collaborates with Bax protein to form pores in the mitochondrial membrane releasing cytochrome c and other apoptotic factors. This process initiates the cascade of caspases leading to programmed cell death.

Pathways

Bak is a critical component of the intrinsic apoptotic pathway. It directly interacts with pro-apoptotic proteins like Bax and anti-apoptotic proteins such as Bcl-2 and Bcl-xL. Bak's action in the apoptotic pathway involves a delicate balance between survival and death signals affecting cellular homeostasis. In addition Bak's interaction with the p53 pathway emphasizes its role in response to DNA damage ensuring damaged cells do not proliferate uncontrollably.

Malfunction or dysregulation of Bak can lead to cancer and neurodegenerative disorders. In cancer reduced Bak activity may allow cells to evade apoptosis promoting tumor survival and growth. Conversely in diseases like Parkinson's excessive Bak activity may result in enhanced neuronal apoptosis. Bak's interaction with proteins such as Bcl-2 also makes it a potential target for therapies aimed at restoring apoptotic balance in affected cells.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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Target data

See full target information BAK1
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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.
For full details, please see our Terms & Conditions

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