Product datasheet

Anti-160 kD Neurofilament Medium antibody [EPR23510-76]
ab254348
リコンビナント

2 References 画像数 22
製品の概要
製品名

Anti-160 kD Neurofilament Medium antibody [EPR23510-76]

製品の詳細

Rabbit monoclonal [EPR23510-76] to 160 kD Neurofilament Medium

由来種

Rabbitアプリケーション

適用あり : IHC-P, Flow Cyt (Intra), WB, IP, ELISA, IHC-Fr, ICC/IF

種交差性

交差種 : Mouse, Rat, Human

免疫原

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

ポジティブ･コントロール

WB: Human cerebellum and nerve tissue lysate. Mouse and rat brain and cerebellum tissue
lysate. Wild-type HEK-293T lysate. IHC-P: Human cerebellum and cerebrum tissue. Mouse andrat cerebellum tissue. IHC-Fr: Mouse and rat cerebrum tissue. ICC/IF: Parental HEK-293T cells.
Mouse and rat primary neural/glia cells. Flow Cyt(Intra): Wild-type HEK-293T cells. Mouse and rat
primary neuron cells. IP: Mouse and rat whole cell lysate.

特記事項

This product is a recombinant monoclonal antibody, which offers several advantages including:- High batch-to-batch consistency and reproducibility- Improved sensitivity and specificity- Long-term security of supply- Animal-free production
For more information see here.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit
monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

製品の特性
製品の状態

Liquid

保存方法

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C longterm. Avoid freeze / thaw cycle.

バッファー

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

精製度

Protein A purified
1

ポリ /モノ

モノクローナル

クローン名

EPR23510-76

アイソタイプ

IgG

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ab254348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

IHC-P

1/4000. Perform heat mediated antigen retrieval with Tris/EDTA
buffer pH 9.0 before commencing with IHC staining protocol.

Flow Cyt (Intra)

1/500.

WB

1/1000. Predicted molecular weight: 102 kDa.

IP

1/30.

ELISA

Use a concentration of 0.125 µg/ml.

IHC-Fr1/100.
Heat mediated antigen retrieval using sodium citrate buffer(10mM citrate pH 6.0 + 0.05% Tween-20)

ICC/IF

1/50.

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 theserines in this motif. It is thought that phosphorylation of NFM results in the formation ofinterfilament 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 andcoincidentally with a change in the neurofilament function.
Phosphorylated in the head and rod regions by the PKC kinase PKN1, leading to the inhibition ofpolymerization.

画像2

All lanes : Anti-160 kD Neurofilament Medium antibody[EPR23510-76] (ab254348) at 1/1000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human nerve tissue lysate
Lane 3 : Human liver tissue lysate
Lysates/proteins at 20 µg per lane.

SecondaryAll lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at
Western blot - Anti-160 kD Neurofilament Medium

1/1000 dilution

antibody [EPR23510-76] (ab254348)

Predicted band size: 102 kDa
Observed band size: 160 kDa

Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The molecular weight is consistent to what has been described in
the literature (PMID:19239416, PMID:27000625).

Negative control: liver (PMID:30541916).
Exposure time: 10 seconds.

Immunohistochemical analysis of paraffin-embedded humancerebellum tissue labelling 160 kD Neurofilament Medium with
ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use
LeicaDS9800 (Bond™ Polymer Refine Detection). The section was
incubated with ab254348 for 30 mins at room temperature. The
immunostaining was performed on a Leica Biosystems BOND® RX
instrument. Counterstained with Hematoxylin.
Positive staining on human cerebellum.
Secondary antibody only control: Secondary antibody is ready touse LeicaDS9800 (Bond™ Polymer Refine Detection).
Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-160 kD Neurofilament

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0,
epitope retrieval solution2) for 20 mins.

Medium antibody (ab254348)

3

Immunohistochemical analysis of paraffin-embedded mouse livertissue labelling 160 kD Neurofilament Medium with ab254348 at
1/4000 (0.111 µg/ml) dilution followed by ready to use
LeicaDS9800 (Bond™ Polymer Refine Detection). The section was
incubated with ab254348 for 30 mins at room temperature. The
immunostaining was performed on a Leica Biosystems BOND® RX
instrument. Counterstained with Hematoxylin.

Negative control: No staining on mouse liver.
Secondary antibody only control: Secondary antibody is ready touse LeicaDS9800 (Bond™ Polymer Refine Detection).
Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-160 kD Neurofilament

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0,
epitope retrieval solution2) for 20 mins.

Medium antibody (ab254348)

Immunofluorescent analysis of parental HEK-293T (EDWT04) andNEFM KO HEK-293T (ED040746) cells labelling 160 kD
Neurofilament Medium with ab254348 at 1/50 (8.86 µg/ml) dilution,
followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor®
488) antibody at 1/1000 dilution (Green). ab195889 Anti-alphaTubulin mouse monoclonal antibody - Microtubule Marker (Alexa
Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red).
The nuclear counterstain was (Blue).
Confocal image showing cytoplasmic staining in parental HEK293T cells and no staining in NEFM KO HEK-293T cells.
Secondary antibody only control: Secondary antibody is ab150077
Immunocytochemistry/ Immunofluorescence - Anti-

Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

160 kD Neurofilament Medium antibody [EPR2351076] (ab254348)

4

Immunofluorescent analysis of mouse primary neural/glia cells fixedwith 4% Paraformaldehyde and permeabilised with 0.1% TritonX100, labelling 160 kD Neurofilament Medium with ab254348 at 1/50
(8.86 µg/ml) dilution. ab11267 Anti-MAP2 mouse monoclonal
antibody was used to counterstain at 1/500 (4 μg/ml). Followed bysecondary ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)
antibody at 1/1000 (2 μg/ml) (Green). ab150120 Goat Anti-Mouse
IgG H&L (Alexa Fluor® 594) was used as secondary counterstain at
1/1000 (2 μg/ml) (Red). The nuclear counterstain was DAPI (Blue).
Confocal image showing cytoplasmic staining in mouse primaryneuron cells.
Immunocytochemistry/ Immunofluorescence - Anti-

Confocal scanning Z step was set as 0.3 μm followed by image

160 kD Neurofilament Medium antibody [EPR23510-

processing with maximum Z projection.

76] (ab254348)

Negative Control 1: ab150120 1/1000 (2 μg/ml)
Negative Control 2: ab11267 1/500 (4 μg/ml),
secondary: ab150077 1/1000 (2μg/ml)

Immunofluorescent analysis of rat primary neural/glia cells fixed with
4% Paraformaldehyde and permeabilised with 0.1% TritonX-100,
labelling 160 kD Neurofilament Medium with ab254348 at 1/50
(8.86 µg/ml) dilution. ab11267 Anti-MAP2 mouse monoclonal
antibody was used to counterstain at 1/500 (4 μg/ml). Followed bysecondary ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)
antibody at 1/1000 (2 μg/ml) (Green). ab150120 Goat Anti-Mouse
IgG H&L (Alexa Fluor® 594) was used as secondary counterstain at
1/1000 (2 μg/ml) (Red). The nuclear counterstain was DAPI (Blue).
Confocal image showing cytoplasmic staining in rat primary neuron
cells.
Immunocytochemistry/ Immunofluorescence - Anti-

Confocal scanning Z step was set as 0.3 μm followed by image

160 kD Neurofilament Medium antibody [EPR23510-

processing with maximum Z projection.

76] (ab254348)

Negative Control 1: ab150120 1/1000 (2 μg/ml)
Negative Control 2: ab11267 1/500 (4 μg/ml), secondary:ab150077 1/1000 (2 μg/ml)

5Flow cytometric analysis of 4% paraformaldehyde fixed 90%
methanol permeabilized wild-type HEK-293T (human embryonickidney epithelial cell, Right)/ 160 kD Neurofilament Medium
knockout HEK-293T cells (Left) labelling 160 kD Neurofilament
Medium with ab254348 at 1/500 dilution (0.1 µg). A Goat anti rabbit
IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as
the secondary antibody.
Positive staining on human wild-type HEK-293T cell line
(ab255449), while no staining on human NEFM knockout HEK293T cells (ab266741).
Flow Cytometry (Intracellular) - Anti-160 kD
Neurofilament Medium antibody [EPR23510-76](ab254348)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100
permeabilized frozen mouse cerebrum tissue labeling 160 kD
Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution
followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor®
488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI(Blue).
Positive staining on mouse cerebrum is observed.
Secondary antibody control: Secondary antibody is ab150077 Goat
Anti-Rabbit IgG H&L (Alexa Fluor® 488 )at 1/1000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10mM
Immunohistochemistry (Frozen sections) - Anti-160

citrate pH 6.0 + 0.05% Tween-20).

kD Neurofilament Medium antibody (ab254348)

6

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100
permeabilized frozen mouse liver tissue labeling 160 kD
Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution
followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor®
488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI
(blue).

Negative control: No staining on mouse liver (PMID: 30541916) isobserved.
Secondary antibody control: Secondary antibody is ab150077 Goat
Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
Immunohistochemistry (Frozen sections) - Anti-160

Heat mediated antigen retrieval using sodium citrate buffer (10mM

kD Neurofilament Medium antibody (ab254348)

citrate pH 6.0 + 0.05% Tween-20).

160 kD Neurofilament Medium was immunoprecipitated from 0.35
mg mouse brain tissue lysate 10 µg with ab254348 at 1/30 dilution(2 µg in 0.35 mg lysates). Western blot was performed on the
immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for
IP secondary antibody (HRP) (ab131366) was used at 1/5000
dilution.

Lane 1: Mouse brain tissue lysate 10 µg.
Lane 2: ab254348 IP in mouse brain tissue lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254348
in mouse brain tissue lysate.
Immunoprecipitation - Anti-160 kD Neurofilament

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Medium antibody (ab254348)

Exposure time: 3.25 secs.

7

ELISA analysis using ab254348 at a range of 0-1000 ng/ml
followed by a Alkaline Phosphatase-conjugated AffiniPure Goat
Anti-Rabbit IgG (H+L）at 1/2500 dilution. Antigen concentration:
1000 ng/ml.
Antigens: Mouse 160 kD Neurofilament Medium.

ELISA - Anti-160 kD Neurofilament Medium antibody[EPR23510-76] (ab254348)

All lanes : Anti-160 kD Neurofilament Medium antibody[EPR23510-76] (ab254348) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse cerebellum tissue lysate
Lane 3 : Mouse liver tissue lysate
Lane 4 : Rat brain tissue lysate
Lane 5 : Rat cerebellum tissue lysate
Lane 6 : Rat liver tissue lysate
Lysates/proteins at 20 µg per lane.
Western blot - Anti-160 kD Neurofilament Medium
antibody [EPR23510-76] (ab254348)

SecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at
1/100000 dilution

Predicted band size: 102 kDa
Observed band size: 160 kDa

8Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The molecular weight is consistent to what has been described in
the literature (PMID:19239416, PMID:27000625).

Negative control: liver (PMID:30541916).
Exposure times: Lane 1-3: 3.25 secs; Lane 4-6: 10 secs.

All lanes : Anti-160 kD Neurofilament Medium antibody[EPR23510-76] (ab254348) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (human embryonic kidney epithelialcell) whole cell lysate

Lane 2 : NEFM knockout HEK-293T whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell) whole celllysateLysates/proteins at 20 µg per lane.

Western blot - Anti-160 kD Neurofilament Medium

Secondaryantibody [EPR23510-76] (ab254348)

All lanes : Goat anti-Mouse IgG H&L (IRDye® 680RD)
preadsorbed (ab216776) at 1/10000 dilution

Predicted band size: 102 kDa
Observed band size: 160 kDa

Blocking and diluting buffer and concentration: Intercept® (TBS)Blocking Buffer diluted with an equal volume of 0.1% TBS.
Lanes 1-3: Merged signal (red and green). Green - ab254348
observed at 160 kDa. Red - loading control ab8245 observed at 36
kDa.
ab254348 Anti-NEFM antibody [EPR23510-76] was shown tospecifically react with NEFM in wild-type HEK-293T cells. Loss of
signal was observed when the knockout cell line ab266741(knockout cell lysate ab257103) was used. Wild-type and NEFM
knockout samples were subjected to SDS-PAGE. ab254348 andAnti-GAPDH antibody [6C5] - Loading Control (ab8245) wereincubated at at 4°C overnight at 1 in 1000 dilution and 1 in 20000
dilution respectively. Blots were developed with Goat anti-Rabbit
IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat
anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
secondary antibodies at 1 in 10000 dilution for 1 hour at room9

temperature before imaging.

Immunohistochemical analysis of paraffin-embedded humancerebrum tissue labelling 160 kD Neurofilament Medium with
ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use
LeicaDS9800 (Bond™ Polymer Refine Detection). The section was
incubated with ab254348 for 30 mins at room temperature. The
immunostaining was performed on a Leica Biosystems BOND® RX
instrument. Counterstained with Hematoxylin.
Positive staining on human cerebrum.
Secondary antibody only control: Secondary antibody is ready touse LeicaDS9800 (Bond™ Polymer Refine Detection).
Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-160 kD Neurofilament

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0,
epitope retrieval solution2) for 20 mins.

Medium antibody (ab254348)

Immunohistochemical analysis of paraffin-embedded
mouse cerebellum tissue labelling 160 kD Neurofilament Medium
with ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready touse LeicaDS9800 (Bond™ Polymer Refine Detection). The section
was incubated with ab254348 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems
BOND® RX instrument. Counterstained with Hematoxylin.
Positive staining on mouse cerebellum.
Secondary antibody only control: Secondary antibody is ready touse LeicaDS9800 (Bond™ Polymer Refine Detection).
Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-160 kD Neurofilament

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0,
epitope retrieval solution2) for 20 mins.

Medium antibody (ab254348)

10

Immunohistochemical analysis of paraffin-embedded rat cerebellum
tissue labelling 160 kD Neurofilament Medium with ab254348 at
1/4000 (0.111 µg/ml) dilution followed by ready to use
LeicaDS9800 (Bond™ Polymer Refine Detection). The section was
incubated with ab254348 for 30 mins at room temperature. The
immunostaining was performed on a Leica Biosystems BOND® RX
instrument. Counterstained with Hematoxylin.
Positive staining on rat cerebellum.
Secondary antibody only control: Secondary antibody is ready touse LeicaDS9800 (Bond™ Polymer Refine Detection).
Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-160 kD Neurofilament

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0,
epitope retrieval solution2) for 20 mins.

Medium antibody (ab254348)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100
permeabilized frozen rat cerebrum tissue labeling 160 kD
Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution
followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa
Fluor® 488) at 1/1000 dilution (Green). The nuclear counterstain
was DAPI (Blue).
Positive staining on rat cerebrum is observed.
Secondary antibody control: Secondary antibody is ab150077 Goat
Anti-Rabbit IgG H&L (Alexa Fluor® 488 )at 1/1000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10mM
Immunohistochemistry (Frozen sections) - Anti-160

citrate pH 6.0 + 0.05% Tween-20).

kD Neurofilament Medium antibody (ab254348)

11

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100
permeabilized frozen rat liver tissue labeling 160 kD Neurofilament
Medium with ab254348 at 1/100 (4.43 µg/ml) dilution followed byab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000
dilution (Green). The nuclear counterstain was DAPI (blue).

Negative control: No staining on rat liver (PMID: 30541916) isobserved.
Secondary antibody control: Secondary antibody is ab150077 Goat
Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10mM
Immunohistochemistry (Frozen sections) - Anti-160

citrate pH 6.0 + 0.05% Tween-20).

kD Neurofilament Medium antibody (ab254348)

Flow cytometric analysis of 4% paraformaldehyde fixed, 90%
methanol permeabilized mouse primary neuron cells labelling 160
kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 µg)/
Right compared with a Rabbit monoclonal IgG isotype control(ab172730) / Left.
A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000
dilution was used as the secondary antibody.

Flow Cytometry (Intracellular) - Anti-160 kD
Neurofilament Medium antibody [EPR23510-76](ab254348)

12

Flow cytometric analysis of 4% paraformaldehyde fixed, 90%
methanol permeabilized rat primary neuron cells cells labelling 160
kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 µg)/
Right compared with a Rabbit monoclonal IgG isotype control(ab172730) / Left.
A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000
dilution was used as the secondary antibody.

Flow Cytometry (Intracellular) - Anti-160 kD
Neurofilament Medium antibody [EPR23510-76](ab254348)

160 kD Neurofilament Medium was immunoprecipitated from 0.35
mg rat brain tissue lysate 10 µg with ab254348 at 1/30 dilution(2 µg in 0.35 mg lysates). Western blot was performed on the
immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for
IP secondary antibody (HRP) (ab131366) was used at 1/5000
dilution.

Lane 1: Rat brain tissue lysate 10 µg.
Lane 2: ab254348 IP in rat brain tissue lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254348
in rat brain tissue lysate.
Immunoprecipitation - Anti-160 kD Neurofilament

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Medium antibody (ab254348)

Exposure time: 5.5 secs.

13

Anti-160 kD Neurofilament Medium antibody(ab254348)

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