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AB266741

Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line

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NEFM KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
4 Images
Western blot - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)
  • WB

Unknown

Western blot - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)

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° 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.

All lanes:

Western blot - Anti-160 kD Neurofilament Medium antibody [NF-09] - Neuronal Marker (<a href='/en-us/products/primary-antibodies/160-kd-neurofilament-medium-antibody-nf-09-neuronal-marker-ab7794'>ab7794</a>) 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 at 20 µg

Lane 2:

NEFM knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 2:

Western blot - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (ab266741)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 102 kDa

Observed band size: 150 kDa

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Sanger Sequencing - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)
  • Sanger seq

Unknown

Sanger Sequencing - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)

Allele-1 : 1 bp insertion in exon 2

Sanger Sequencing - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)
  • Sanger seq

Unknown

Sanger Sequencing - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)

Allele-2 : Insertion of the selection cassette in exon 2.

Cell Culture - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)
  • Cell Culture

Lab

Cell Culture - Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line (AB266741)

Representative images NEFM 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 a EVOS M5000 microscope.

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, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2

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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
NEFM
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
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.

The 160 kD Neurofilament Medium also known as NEFM NF-M or NEF-M belongs to the family of neurofilament proteins. This protein is an important structural component of neurons particularly in the axonal cytoskeleton. Neurofilament Medium has a molecular mass of around 160 kilodaltons (kD). It shows high levels of expression in neuronal tissues contributing to the maintenance of axonal caliber. Commonly research recognizes it alongside other neurofilament proteins emphasizing its role in providing structural integrity to neurons.
Biological function summary

Neurofilament Medium participates in the formation of a stable network of neurofilaments within neurons. Acting as a part of the intermediate filament protein family it forms a complex with other neurofilaments such as NF-L and NF-H. This complex supports neuron structure and plays an important role in axonal transport. The heteropolymeric nature of neurofilaments contributes to their mechanical stability facilitating essential neuronal functions by aligning neurofilaments along the axon.

Pathways

Neurofilament Medium integrates into the cytoskeletal arrangement pathways that govern axonal transport and stability. It plays a significant role in the neuronal transport pathway associating with other proteins such as dynein and kinesin responsible for the motor functions along axons. Also it relates to the MAP kinase pathway where phosphorylation events modulate its assembly and disassembly dynamics in response to cellular signals.

Neurofilament Medium closely relates to neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Aberrant phosphorylation or aggregation of NEFM can disrupt neuronal function leading to pathology. In ALS the abnormal accumulation of neurofilaments correlates with motor neuron degeneration. In Alzheimer's its interaction with tau protein poses significant interest as altered states of either could exacerbate neurofibrillary tangles worsening cognitive decline.
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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 NEFM
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Product promise

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For full details, please see our Terms & Conditions

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