Human SDHB knockout HEK-293 cell line (ab260860)
Overview
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Product name
Human SDHB knockout HEK-293 cell line -
Parental Cell Line
HEK-293 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, ICCmore 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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
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
Iron-sulfur protein (IP) subunit of succinate dehydrogenase (SDH) that is involved in complex II of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone (coenzyme Q). -
Pathway
Carbohydrate metabolism; tricarboxylic acid cycle; fumarate from succinate (eukaryal route): step 1/1. -
Involvement in disease
Defects in SDHB are a cause of susceptibility to pheochromocytoma (PCC) [MIM:171300]. A catecholamine-producing tumor of chromaffin tissue of the adrenal medulla or sympathetic paraganglia. The cardinal symptom, reflecting the increased secretion of epinephrine and norepinephrine, is hypertension, which may be persistent or intermittent.
Defects in SDHB are the cause of hereditary paragangliomas type 4 (PGL4) [MIM:115310]; also known as familial non-chromaffin paragangliomas type 4. Paragangliomas refer to rare and mostly benign tumors that arise from any component of the neuroendocrine system. PGL4 is characterized by the development of mostly benign, highly vascular, slow growing tumors in the head and neck. In the head and neck region, the carotid body is the largest of all paraganglia and is also the most common site of the tumors.
Defects in SDHB are a cause of paraganglioma and gastric stromal sarcoma (PGGSS) [MIM:606864]; also called Carney-Stratakis syndrome. Gastrointestinal stromal tumors may be sporadic or inherited in an autosomal dominant manner, alone or as a component of a syndrome associated with other tumors, such as in the context of neurofibromatosis type 1 (NF1). Patients have both gastrointestinal stromal tumors and paragangliomas. Susceptibility to the tumors was inherited in an apparently autosomal dominant manner, with incomplete penetrance.
Defects in SDHB are a cause of Cowden-like syndrome (CWDLS) [MIM:612359]. Cowden-like syndrome is a cancer predisposition syndrome associated with elevated risk for tumors of the breast, thyroid, kidney and uterus. -
Sequence similarities
Belongs to the succinate dehydrogenase/fumarate reductase iron-sulfur protein family.
Contains 1 2Fe-2S ferredoxin-type domain.
Contains 1 4Fe-4S ferredoxin-type domain. -
Cellular localization
Mitochondrion inner membrane. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
- Anti-SDHB antibody [21A11AE7] (ab14714)
- Anti-SDHB antibody (ab151684)
- Anti-SDHB antibody [EPR10880] (ab175225)
- Anti-SDHB antibody [EPR13042(B)] (ab178423)
- Alexa Fluor® 647 Anti-SDHB antibody [21A11AE7] (ab197722)
- Alexa Fluor® 488 Anti-SDHB antibody [21A11AE7] (ab197902)
- HRP Anti-SDHB antibody [21A11AE7] (ab197903)
- Anti-Lin28B antibody [EPR18717] - BSA and Azide free (ab240326)
- Anti-alpha smooth muscle Actin antibody [1A4] - BSA and Azide free (ab240654)
- Anti-SDHB antibody [EPR10880] - BSA and Azide free (ab249876)
- Anti-SDHB antibody [EPR13042(B)] - BSA and Azide free (ab250046)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab260860 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration.
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| ICC |
Use at an assay dependent concentration.
|
| Notes |
|---|
|
WB
Use at an assay dependent concentration. |
|
ICC
Use at an assay dependent concentration. |
Images
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Western blot - Human SDHB knockout HEK-293 cell line (ab260860)All lanes : HRP Anti-SDHB antibody [21A11AE7] (ab197903) at 1/5000 dilution
Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : SDHB knockout HEK-293 whole cell lysate
Lane 3 : Hep G2 whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab197903 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.
ab197903 was shown to specifically react with SDHB in wild-type HEK-293 cells as signal was lost in SDHB knockout cell line ab260860 (knockout cell lysate ab261652). Wild-type and SDHB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab197903 and ab130007 (Mouse anti-Vinculin 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.
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Immunocytochemistry/ Immunofluorescence - Human SDHB knockout HEK-293 cell line (ab260860)ab197722 staining SDHB in wild-type HEK-293 cells (top panel) and SDHB knockout HEK-293 cells (ab260860) (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 ab197722 at 1/500 dilution (shown in red) and ab195887 (Mouse monoclonal to alpha Tubulin - Alexa Fluor® 488) at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). -
Western blot - Human SDHB knockout HEK-293 cell line (ab260860)All lanes : Anti-SDHB antibody (ab151684) at 1 µg/ml
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : SDHB knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab151684 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.
ab151684 was shown to recognize SDHB in wild-type HEK-293 cells as signal was lost at the expected MW in SDHB knockout cell line ab260860 (knockout cell lysate ab261652). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and SDHB knockout samples were subjected to SDS-PAGE. Ab151684 and ab130007 (Mouse anti-Vinculin 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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Western blot - Human SDHB knockout HEK-293 cell line (ab260860)All lanes : Anti-SDHB antibody [EPR13042(B)] (ab178423) at 1/5000 dilution
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : SDHB knockout HEK-293 whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab178423 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.
ab178423 was shown to recognize SDHB in wild-type HEK-293 cells as signal was lost at the expected MW in SDHB knockout cell line ab260860 (knockout cell lysate ab261652). Additional cross-reactive bands were observed in the wild-type and knockout cell lysate. Wild-type and SDHB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab178423 and ab130007 (Mouse anti Vinculin 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.
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Western blot - Human SDHB knockout HEK-293 cell line (ab260860)All lanes : Anti-SDHB antibody [EPR10880] (ab175225) at 1/50000 dilution
Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : SDHB knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab175225 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.
ab175225 was shown to recognize SDHB in wild-type HEK-293 cells as signal was lost at the expected MW in SDHB knockout cell line ab260860 (knockout cell lysate ab261652). Additional cross-reactive bands were observed in the wild-type and knockout cell lysate. Wild-type and SDHB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab175225 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/50000 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.
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Immunocytochemistry/ Immunofluorescence - Human SDHB knockout HEK-293 cell line (ab260860)ab197902 staining SDHB in wild-type HEK-293 cells (top panel) and SDHB knockout HEK-293 cells (ab260860) (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 ab197902 at 1/500 dilution (shown in green) and ab190573 (Rabbit monoclonal to alpha Tubulin - Alexa Fluor® 647) at 1/250 dilution (shown in red) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human SDHB knockout HEK-293 cell line (ab260860)ab14714 staining SDHB in wild-type HEK-293 cells (top panel) and SDHB knockout HEK-293 cells (ab260860) (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 ab14714 at 5µg concentration and ab6046 (Rabbit polyclonal to beta 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 mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab260860 has not yet been referenced specifically in any publications.