重组Anti-160 kD Neurofilament Medium抗体[EPR23510-76] (ab254348)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23510-76] to 160 kD Neurofilament Medium
- Suitable for: IHC-P, Flow Cyt (Intra), WB, IP, ELISA, IHC-Fr, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
概述
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产品名称
Anti-160 kD Neurofilament Medium抗体[EPR23510-76]
参阅全部 160 kD Neurofilament Medium 一抗 -
描述
兔单克隆抗体[EPR23510-76] to 160 kD Neurofilament Medium -
宿主
Rabbit -
经测试应用
适用于: IHC-P, Flow Cyt (Intra), WB, IP, ELISA, IHC-Fr, ICC/IFmore details -
种属反应性
与反应: Mouse, Rat, Human -
免疫原
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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阳性对照
- 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 and rat 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.
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常规说明
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
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
EPR23510-76 -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
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Compatible Secondaries
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Isotype control
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab254348于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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IHC-P |
1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
1/500.
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WB |
1/1000. Predicted molecular weight: 102 kDa.
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IP |
1/30.
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ELISA |
Use a concentration of 0.125 µg/ml.
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IHC-Fr |
1/100.
Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) |
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ICC/IF |
1/50.
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说明 |
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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-Fr
1/100. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) |
ICC/IF
1/50. |
靶标
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功能
Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. -
序列相似性
Belongs to the intermediate filament family. -
翻译后修饰
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
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数据库链接
- Entrez Gene: 4741 Human
- Entrez Gene: 18040 Mouse
- Entrez Gene: 24588 Rat
- Omim: 162250 Human
- SwissProt: P07197 Human
- SwissProt: P08553 Mouse
- SwissProt: P12839 Rat
- Unigene: 458657 Human
see all -
别名
- 150kDa medium antibody
- 160 kDa neurofilament protein antibody
- NEF3 antibody
see all
图片
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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.
Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Predicted band size: 102 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?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.
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Immunohistochemical analysis of paraffin-embedded human 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 human cerebellum.
Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded mouse liver 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.
Negative control: No staining on mouse liver.
Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunofluorescent analysis of parental HEK-293T (EDWT04) and NEFM 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-alpha Tubulin 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 HEK-293T cells and no staining in NEFM KO HEK-293T cells.
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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Immunofluorescent analysis of mouse 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 by secondary 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 primary neuron cells.
Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.Negative Control 1: ab150120 1/1000 (2 μg/ml)
Negative Control 2: ab11267 1/500 (4 μg/ml), secondary: ab150077 1/1000 (2μg/ml)
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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 by secondary 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.
Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.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)
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Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized wild-type HEK-293T (human embryonic kidney 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 HEK-293T cells (ab266741).
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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 citrate pH 6.0 + 0.05% Tween-20).
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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) 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 citrate pH 6.0 + 0.05% Tween-20).
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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.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3.25 secs.
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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.
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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.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 102 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?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.
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All lanes : Anti-160 kD Neurofilament Medium antibody [EPR23510-76] (ab254348) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : NEFM knockout HEK-293T whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
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 why is the actual band size different from the predicted?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 to specifically 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 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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 room temperature before imaging.
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Immunohistochemical analysis of paraffin-embedded human cerebrum 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 to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
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 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 mouse cerebellum.
Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
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 to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
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 citrate pH 6.0 + 0.05% Tween-20).
-
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 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 rat liver (PMID: 30541916) 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 citrate pH 6.0 + 0.05% Tween-20).
-
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.
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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.
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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.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5.5 secs.
实验方案
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
数据表及文件
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SDS download
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Datasheet download
Certificate of Compliance
文献 (2)
ab254348 被引用在 2 文献中.
- McNamara SL et al. Anti-inflammatory therapy enables robot-actuated regeneration of aged muscle. Sci Robot 8:eadd9369 (2023). PubMed: 36947599
- Guo J et al. Blockage of MLKL prevents myelin damage in experimental diabetic neuropathy. Proc Natl Acad Sci U S A 119:e2121552119 (2022). PubMed: 35344427