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AB266321

Human CSTB (Cystatin-B) knockout HEK-293T cell line

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CSTB KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 1. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
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Western blot - Human CSTB (Cystatin-B) knockout HEK-293T cell line (AB266321)
  • WB

Lab

Western blot - Human CSTB (Cystatin-B) knockout HEK-293T cell line (AB266321)

Lanes 1-4 : Merged signal (red and green). Green - ab92449 observed at 15 kDa. Red - loading control ab8245 observed at 36 kDa.

ab92449 Anti-Cystatin-B antibody [EPR3931] was shown to specifically react with Cystatin-B in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266321 (knockout cell lysate ab257905) was used. Wild-type and Cystatin-B knockout samples were subjected to SDS-PAGE. ab92449 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° 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.

All lanes:

Western blot - Anti-Cystatin-B antibody [EPR3931] (<a href='/en-us/products/primary-antibodies/cystatin-b-antibody-epr3931-ab92449'>ab92449</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

CSTB knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human CSTB (Cystatin-B) knockout HEK-293T cell line (ab266321)

Lane 3:

U87-MG cell lysate at 20 µg

Lane 4:

JAR cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 11 kDa

Observed band size: 15 kDa

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Cell Culture - Human CSTB (Cystatin-B) knockout HEK-293T cell line (AB266321)
  • Cell Culture

Unknown

Cell Culture - Human CSTB (Cystatin-B) knockout HEK-293T cell line (AB266321)

Representative images of CSTB knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.

Sanger Sequencing - Human CSTB (Cystatin-B) knockout HEK-293T cell line (AB266321)
  • Sanger seq

Unknown

Sanger Sequencing - Human CSTB (Cystatin-B) knockout HEK-293T cell line (AB266321)

Homozygous : Insertion of the selection cassette in exon 1

Key facts

Cell type

HEK-293T

Species or organism

Human

Tissue

Kidney

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 1

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Complementary Products

Primary Antibodies

AB92449

Anti-Cystatin-B antibody [EPR3931]

RabMAb|Recombinant|KO Validated

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cystatin-B antibody [EPR3931] (AB92449)

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Cell Lines & Lysates

AB266257

Human VRK1 knockout HEK-293T cell line

Advanced Validation

Western blot - Human VRK1 knockout HEK-293T cell line (AB266257)

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Cell Lines & Lysates

AB266343

Human RAD23A (hHR23A) knockout HEK-293T cell line

Advanced Validation

Western blot - Human RAD23A (hHR23A) knockout HEK-293T cell line (AB266343)

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Cell Lines & Lysates

AB266323

Human PDCD6 (ALG-2) knockout HEK-293T cell line

Advanced Validation

Western blot - Human PDCD6 (ALG-2) knockout HEK-293T cell line (AB266323)

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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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
CSTB
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
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.

Cystatin-B also known as STEFIN-B is a protease inhibitor with a molecular mass of approximately 11 kilodaltons. It belongs to the family of cystatins which are expressed widely in human tissues with notable presence in the brain liver and kidneys. Cystatin-B acts by inhibiting cysteine proteases preventing them from degrading proteins indiscriminately. The inhibition is important for maintaining cellular protein balance and integrity.
Biological function summary

Cysteine protease inhibition by Cystatin-B protects cells from uncontrolled proteolysis which can lead to tissue damage. This protein plays an important role in the regulation of various cellular processes including apoptosis phagocytosis and the response to oxidative stress. It does not form part of a larger complex but interacts directly with its target proteases to exert its inhibitory effects.

Pathways

Cystatin-B influences proteolytic pathways by modulating the activity of cysteine proteases such as cathepsins. By inhibiting these enzymes Cystatin-B participates in the regulation of the lysosomal degradation pathway and apoptotic pathways which are important for cellular turnover and programmed cell death. Cathepsins particularly cathepsin B and L are related proteins that are significantly modulated through their interaction with Cystatin-B.

Mutations or dysregulation of Cystatin-B are associated with progressive myoclonus epilepsy and certain neurodegenerative diseases. The lack of functional Cystatin-B leads to excessive activity of proteases like cathepsin B resulting in increased proteolysis and neuronal damage. In some cases this can also connect to disorders involving oxidative stress exacerbating the detrimental cycle of cellular injury.
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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 CSTB
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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

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

For licensing inquiries, please contact partnerships@abcam.com