Recombinant Anti-Clathrin light chain antibody [EPR24231-72] (ab271185)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR24231-72] to Clathrin light chain
- Suitable for: IHC-P, Flow Cyt (Intra), WB, IP, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Clathrin light chain antibody [EPR24231-72]
See all Clathrin light chain primary antibodies -
Description
Rabbit monoclonal [EPR24231-72] to Clathrin light chain -
Host species
Rabbit -
Tested applications
Suitable for: IHC-P, Flow Cyt (Intra), WB, IP, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa, HEK-293, NCI-H1299, MEF, C2C12, PC-12 and C6 whole cell lysartes. Human Clathrin light chain recombinant protein. IHC-P: Human cerebrum tissue; Mouse cerebrum tissue; Rat cerebrum tissue. ICC/IF: MEF, HeLa and C2C12 cells. Flow Cyt-intra: HeLa and C2C12 cells. IP: HeLa and MEF whole cell lysates.
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General notes
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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
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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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR24231-72 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab271185 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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IHC-P |
1/5000. 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. Detects a band of approximately 35 kDa (predicted molecular weight: 27 kDa).
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IP |
1/30.
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ICC/IF |
1/250.
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Notes |
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IHC-P
1/5000. 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. Detects a band of approximately 35 kDa (predicted molecular weight: 27 kDa). |
IP
1/30. |
ICC/IF
1/250. |
Target
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Function
Clathrin is the major protein of the polyhedral coat of coated pits and vesicles. -
Cellular localization
Cytoplasmic vesicle membrane. Membrane > coated pit. Cytoplasmic face of coated pits and vesicles. - Information by UniProt
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Database links
- Entrez Gene: 1211 Human
- Entrez Gene: 1212 Human
- Entrez Gene: 12757 Mouse
- Entrez Gene: 74325 Mouse
- Entrez Gene: 116561 Rat
- Entrez Gene: 83800 Rat
- Omim: 118960 Human
- Omim: 118970 Human
see all -
Alternative names
- Clathrin light chain A antibody
- Clathrin light chain B antibody
- Clathrin light chain LCA antibody
see all
Images
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All lanes : Anti-Clathrin light chain antibody [EPR24231-72] (ab271185) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CLTA knockout HEK-293T cell lysate
Lane 3 : A431 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 27 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Clathrin light chain antibody [EPR24231-72] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271185 was shown to bind specifically to Clathrin light chain. A band was observed at 35 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in CLTA knockout cell line ab267334 (knockout cell lysate ab258366). To generate this image, wild-type and CLTA knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling Clathrin light chain with ab271185 at 1/5000 (0.102 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining in human cerebrum. The section was incubated with ab271185 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a 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 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF cells labelling Clathrin light chain with ab271185 at 1/250 (2.036 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in MEF cells is observed. 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 DAPI (Blue).
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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Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (Human cervix adenocarcinoma cell) cells labelling Clathrin light chain with ab271185 at 1/500 dilution (0.1ug)(Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
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All lanes : Anti-Clathrin light chain antibody [EPR24231-72] (ab271185) at 1/1000 dilution
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate
Lane 4 : MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate
Lane 5 : C2C12 (mouse myoblasts myoblast) whole cell lysate
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 7 : C6 (rat glial tumor glial cell) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 27 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The observed MW is consistent with what has been described in the literature (PMID: 31672988, 24852344).
Exposure time: 70 seconds.
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Clathrin light chain was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab271185 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271185 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug
Lane 2: ab271185 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271185 in HeLa whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds
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Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling Clathrin light chain with ab271185 at 1/5000 (0.102 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining in mouse cerebrum. The section was incubated with ab271185 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a 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 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling Clathrin light chain with ab271185 at 1/250 (2.036 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HeLa cells is observed. 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 DAPI (Blue).
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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All lanes : Anti-Clathrin light chain antibody [EPR24231-72] (ab271185) at 1/1000 dilution
Lane 1 : Human Clathrin light chain recombinant protein
Lane 2 : Human CLTB recombinant protein
Lysates/proteins at 0.01 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 27 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
This antibody does not recognize human CLTB.
Exposure time: 70 seconds.
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Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling Clathrin light chain with ab271185 at 1/5000 (0.102 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining in rat cerebrum. The section was incubated with ab271185 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a 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 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C2C12 cells labelling Clathrin light chain with ab271185 at 1/250 (2.036 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in C2C12 cells is observed. 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 DAPI (Blue).
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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Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized C2C12 (Mouse myoblasts myoblast) cells labelling Clathrin light chain with ab271185 at 1/500 dilution (0.1ug)(Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
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Clathrin light chain was immunoprecipitated from 0.35 mg MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate with ab271185 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271185 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate 10 ug
Lane 2: ab271185 IP in MEF whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271185 in MEF whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds
Protocols
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.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (0)
ab271185 has not yet been referenced specifically in any publications.