LAMP2 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 1.
Images
Key facts
- Cell type
HeLa
- Species or organism
Human
- Tissue
Cervix
- Form
Liquid
- Knockout validation
Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 1
Alternative names
CD107 antigen-like family member B, CD107b, LAMP 2C, LAMP2_HUMAN, LAMPB, LGP 96, LGP110, Lysosomal associated membrane protein 2, Lysosome-associated membrane glycoprotein 2, Lysosome-associated membrane protein 2, MAC3
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LAMP2 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 1.
Key facts
- Cell type
HeLa
- Species or organism
Human
- Tissue
Cervix
- Form
Liquid
- Knockout validation
Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 1
- Disease
Adenocarcinoma
- Concentration
Properties
- Gene name
LAMP2
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Sanger Sequencing, Western blot
- Zygosity
Homozygous
Quality control
- STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
EU: 2 US: 2
- Adherent/suspension
Adherent
- Gender
Female
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.
Storage
- Shipped at conditions
Dry Ice
- Appropriate short-term storage conditions
-196°C
- Appropriate long-term storage conditions
-196°C
Notes
Recommended control: Human wild-type HeLa cell line (Human wild-type HeLa cell line ab255448). 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.
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.
Supplementary info
This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
LAMP2 also known as CD107b is an important protein found in the lysosomal membrane. It plays a significant role in lysosome function facilitating autophagy and the degradation of cellular debris. LAMP2 proteins are expressed in most tissues especially in tissues with high lysosomal activity like the liver kidney and heart. The LAMP2 molecule has a molecular weight of approximately 45 kDa. Researchers frequently use LAMP2 staining and LAMP2 marker to study its cellular localization and expression levels.
- Biological function summary
LAMP2 is essential for the normal fusion of lysosomes with autophagosomes. This process ensures the recycling of cellular components and maintenance of cellular homeostasis. LAMP2 acts in conjunction with other members of the lysosome-associated membrane protein family LAMP1 and LAMP3 forming part of a larger complex. This interaction is fundamental in managing the degradation pathways within the cell supporting processes such as lipid metabolism and protein turnover.
- Pathways
The activity of LAMP2 is critical in the autophagic and degradation pathways within the cell. It particularly interacts in the autophagy-lysosome pathway where it aids the clearance of damaged organelles and protein aggregates. LAMP2 interaction with proteins such as the autophagy-related protein LC3 helps this degradation process. Additionally LAMP2 plays a role in endocytic pathways linking it to proteins involved in vesicle trafficking and membrane fusion.
- Associated diseases and disorders
Mutations or deficiencies in LAMP2 are associated with Danon disease a type of lysosomal storage disorder. Patients with Danon disease exhibit symptoms such as cardiomyopathy skeletal myopathy and intellectual disabilities. LAMP2 is also implicated in other lysosomal disorders with a connection to the malfunction of autophagy processes. Its dysfunction is often linked with proteins responsible for cellular degradation affecting the normal homeostatic balance and leading to the accumulation of autophagic substrates.
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5 product images
Sanger Sequencing - Human LAMP2 knockout HeLa cell line (ab255402)
Homozygous: Insertion of the selection cassette in exon 1.
Immunocytochemistry/ Immunofluorescence - Human LAMP2 knockout HeLa cell line (ab255402)
Side-by-side comparison of ICC performance using the rabbit polyclonal Anti-LAMP2A antibody - Lysosome Marker ab18528 and RabMab® Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068. Staining was performed on wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Tween-20 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 Anti-LAMP2A antibody - Lysosome Marker ab18528 or Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068 overnight at +4°C at 3 different concentrations: 1.0 μg/mL, 0.2 μg/mL and 0.04 μg/mL. Secondary antibody incubation was at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) (shown in green) at 1/1000 and nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Some cytoplasmic cross-reactivity is seen using Anti-LAMP2A antibody - Lysosome Marker ab18528 at 1.0 μg/mL, but further titration of the antibody improves the ICC staining result. The RabMab® Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068 shows negligiblenon-specific staining across the dilution range. Quantification of the antibody signal was performed using a minimum of 135 cells and data are presented as mean ± SD.Optimal dilutions/concentrations may vary across different cell types/experiment conditions and should be determined by the end user.
Western blot - Human LAMP2 knockout HeLa cell line (ab255402)
False colour image of Western blot: Anti-LAMP2 antibody [EPR19531] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-LAMP2 antibody [EPR19531] ab199947 was shown to bind specifically to LAMP2. A band was observed at 100 kDa in wild-type HeLa cell lysates with no signal observed at this size in LAMP2 knockout cell line ab255402 (knockout cell lysate Human LAMP2 knockout HeLa cell lysate ab263861). The band observed in the knockout lysate lane below 100 kDa is likely to represent a truncated form of LAMP2. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and LAMP2 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
Lane 1: Wild-type HeLa cell at 20 µg
Lane 2: LAMP2 knockout HeLa cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 100 kDa
Western blot - Human LAMP2 knockout HeLa cell line (ab255402)
False colour image of Western blot: Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068 was shown to bind specifically to LAMP2A. A band was observed at 100 kDa in wild-type HeLa cell lysates with no signal observed at this size in LAMP2 knockout cell line ab255402 (knockout cell lysate Human LAMP2 knockout HeLa cell lysate ab263861). The band observed in the knockout lysate lane below 100 kDa is likely to represent a truncated form of LAMP2A. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and LAMP2 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
Lane 1: Wild-type HeLa cell at 20 µg
Lane 2: LAMP2 knockout HeLa cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 100 kDa
Immunocytochemistry/ Immunofluorescence - Human LAMP2 knockout HeLa cell line (ab255402)
Side-by-side comparison of ICC performance using the rabbit polyclonal Anti-LAMP2A antibody - Lysosome Marker ab18528 and RabMab® Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068. Staining was performed on wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Tween-20 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 Anti-LAMP2A antibody - Lysosome Marker ab18528 or Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068 overnight at +4°C at 3 different concentrations: 1.0 μg/mL, 0.2 μg/mL and 0.04 μg/mL. Secondary antibody incubation was at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) (shown in green) at 1/1000 and nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Some cytoplasmic cross-reactivity is seen using Anti-LAMP2A antibody - Lysosome Marker ab18528 at 1.0 μg/mL, but further titration of the antibody improves the ICC staining result. The RabMab® Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker ab125068 shows negligible non-specific staining across the dilution range. Quantification of the antibody signal was performed using a minimum of 180 cells and data are presented as mean ± SD.Optimal dilutions/concentrations may vary across different cell types/experiment conditions and should be determined by the end user.
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