MTMR12 Knockout Hela Cell Line

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LM02200150712

Product ID： LM02200150712

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产品描述
细胞复苏
细胞传代
细胞冻存
抗体验证结果

- Commodity name: MTMR12 Knockout Hela Cell Line
- Commodity ID: LM02200150712
- Gene Symbol: MTMR12 KIAA1682 PIP3AP
- Ensembl ID: ENSG00000150712
- Uniprot ID: Q9C0I1
- 宿主细胞 / 类型: HeLa/Human cervical cancer cells
- NCBI Gene ID: 54545
- 规格: 1×10^6 cells/ cryovials
- 生长培养基: MEM (including NEAA) + 10% FBS + 1% P,S
- 筛选标记: N/A
- 生长特性: Adherent cells, epithelial-like
- 培养条件: Incubator at 37℃, 5% CO2; passage ratio of 1:2 to 1:4
- 倍增时间: ~24-36 hours
- 参考换液频率: 2~3 times/week
- 支原体检测结果: Negative
- 敲除效率（Sanger测序）: 100%
- 蛋白质组验证结果: Protein level verification completed
- 抗体货号: Adding
- 目标基因介绍: Acts as an adapter for myotubularin-related phosphatases (PubMed:11504939, PubMed:12847286, PubMed:23818870). Regulates phosphatase MTM1 protein stability and possibly its intracellular location (PubMed:23818870). By stabilizing MTM1 protein levels, it is required for skeletal muscle maintenance but not for myogenesis (By similarity).
- 细胞开发路径: Generate stable KO Cell Pool using the CRISPR-RNP method; Sanger sequencing results show a 100% knockout efficiency of the KO Cell Pool.
- 应用: Highly efficient gene knockout cell lines (KO Cell Line) are particularly suitable for preliminary functional analysis, the development of complex disease models, precision drug screening, and a wide range of gene discovery research.
Key words:
- MTMR12 KIAA1682 PIP3AP
01. Preheat the complete medium in a 37℃ water bath.
02. Thaw the frozen tube in a 37℃ water bath for 1-2 minutes.
03. Transfer the frozen tube to a biosafety cabinet and wipe the surface with 70% ethanol.
04. Unscrew the cap of the frozen tube and gently transfer the cell suspension to a sterile centrifuge tube containing 9mL of complete medium.
05. Centrifuge at 125g for 5-7 minutes at room temperature, discard the supernatant.
06. Resuspend the cell pellet with 5mL of complete medium and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to an incubator at 37℃, 5% CO2.
08. Reference passage ratio: 1/2 to 1/4 passage, full growth in 2-3 days.
01. When the cell confluence in the culture flask reaches over 80%-90%, the cells can be passaged.
02. Take out the culture medium, PBS, trypsin (0.25% Trypsin_EDTA Gibco 25200-056), etc. from the 4℃ refrigerator, place them in a 37℃ water bath until the temperature is close to 37℃, take them out, spray 75% alcohol on the surface of the bottle, and place them in a biosafety cabinet.

03. Take out the culture flask to be passaged from the incubator, spray the bottle with 75% alcohol, and place it in a biosafety cabinet.
04. To avoid dispersing the cells, rinse the cells along the upper wall of the culture flask with PBS, discard after washing the cells, and add 2mL to T25.
05. Add the corresponding volume of trypsin (1.5mL for T75, 0.5mL for T25), and gently shake the bottle to spread the trypsin evenly over the bottom of the cells. The dosage can be increased or decreased appropriately depending on the actual situation. When most of the cells fall off after about 1-2min, add the corresponding volume of complete culture medium to stop digestion, and gently pipette with a 5mL pipette until all the cells fall off.
06. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge the suspension at 300g for 5min, and discard the supernatant.
07. Transfer 5mL of complete culture medium to resuspend the cells, adjust the inoculation ratio as needed, and supplement the complete culture medium in the culture flask, add to 13-15mL for T75, add to 5mL for T25, and add 1% double antibody.
08. After covering the bottle cap and tightening it, gently shake the bottle to mix the cells evenly, and then place it in a 37℃, 5% CO2 incubator.
01. Prepare freezing solution and pre-chill in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements. Check the following conditions under a microscope: healthy appearance and morphological characteristics, growth cycle (late logarithmic phase), no signs of contamination or degradation.
03. Digest and centrifuge the cells (refer to the subculture procedure for specific steps)
04. Add freezing solution to resuspend the cells according to the amount of 1mL per tube, mix well and aliquot into freezing tubes.
05. Place the cells in a programmed cooling box and freeze in a -80℃ refrigerator.
06. Subsequently, transfer the cells to a liquid nitrogen tank for long-term storage.
Simultaneous Western Blot (WB) analysis of lysates from wild-type and knockout (KO) cells was performed using a specific monoclonal antibody. The target protein band was present in the WT cells but absent or weakened in the KO cells.

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