Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (ab266741)
製品の概要
-
製品名
Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
アプリケーション
適用あり: WBmore details -
Biosafety level
2 -
特記事項
Recommended control: Human wild-type HEK293T cell line (ab255449). 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.
製品の特性
-
Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Kidney -
Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12 -
Mycoplasma free
Yes -
保存方法
Shipped on Dry Ice. Store in liquid nitrogen. -
バッファー
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究分野
ターゲット情報
-
機能
Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. -
配列類似性
Belongs to the intermediate filament family. -
翻訳後修飾
There are a number of repeats of the tripeptide K-S-P, NFM is phosphorylated on a number of the serines in this motif. It is thought that phosphorylation of NFM results in the formation of interfilament cross bridges that are important in the maintenance of axonal caliber.
Phosphorylation seems to play a major role in the functioning of the larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincidentally with a change in the neurofilament function.
Phosphorylated in the head and rod regions by the PKC kinase PKN1, leading to the inhibition of polymerization. - Information by UniProt
関連製品
-
KO cell lysates
-
Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab266741の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
---|---|---|
WB |
Use at an assay dependent concentration. Predicted molecular weight: 102 kDa.
|
特記事項 |
---|
WB
Use at an assay dependent concentration. Predicted molecular weight: 102 kDa. |
画像
-
All lanes : Anti-160 kD Neurofilament Medium antibody [NF-09] - Neuronal Marker (ab7794) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : NEFM knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution
Predicted band size: 102 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab7794 observed at 150 kDa. Red - loading control ab181602 observed at 36 kDa.
ab7794 Anti-160 kD Neurofilament Medium antibody [NF-09] was shown to specifically react with 160 kD Neurofilament Medium in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266741 (knockout cell lysate ab257103) was used. Wild-type and 160 kD Neurofilament Medium knockout samples were subjected to SDS-PAGE. ab7794 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
Allele-1: 1 bp insertion in exon 2
-
Allele-2: Insertion of the selection cassette in exon 2.
プロトコール
データシートおよび資料
-
SDS download
-
Datasheet download
参考文献 (0)
ab266741 は論文での使用が確認できていません。