BD Transduction Laboratories™ Purified Mouse Anti-Clathrin Heavy Chain
Clone 23/Clathrin Heavy Chain
(RUO)
Preparation And Storage
Recommended Assay Procedures
Immunofluorescent staining and bioimaging
Methanol Procedure for a 96 well plate:
Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample.
Triton-X 100 Procedure for a 96 well plate:
Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
- Triton is a trademark of the Dow Chemical Company.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Clathrin is the major protein component in the coat formed around pits and vesicles involved in receptor-mediated endocytosis. Clathrin forms a non-covalently bound triskelion structure composed of three heavy chains (192 kDa each) and three light chains (23-25 kDa each). Each leg of the triskelion structure contains one heavy and one light chain. The three heavy chains forming the triskelion structure are attached at their respective proximal ends like spokes on a wheel. Clathrin heavy chain is composed of a terminal globular domain, a distal segment containing several areas sensitive to enzymatic cleavage, and a proximal segment which contains a light chain binding site. The proximal and distal domains are connected by a joint segment at which there is a sharp bend in the heavy chains of fully-assembled triskelia. Although the calculated molecular weight is 192 kDa, clathrin heavy chain migrates at approximately 180 kDa.
Development References (5)
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Kalthoff C, Groos S, Kohl R, Mahrhold S, Ungewickell EJ. Clint: a novel clathrin-binding ENTH-domain protein at the Golgi. Mol Biol Cell. 2002; 13(11):4060-4073. (Clone-specific: Immunofluorescence, Western blot). View Reference
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Liu SH, Wong ML, Craik CS, Brodsky FM. Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs. Cell. 1995; 83(2):257-267. (Biology). View Reference
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Okamoto CT, Karam SM, Jeng YY, Forte JG, Goldenring JR. Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells. Am J Physiol. 1998; 274(1):C1017-C1029. (Clone-specific: Immunohistochemistry, Western blot). View Reference
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Padilla PI, Chang MJ, Pacheco-Rodriguez G, Adamik R, Moss J, Vaughan M. Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1): effects of FK506. Proc Natl Acad Sci U S A. 2003; 100(5):2322-2327. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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van Kerkhof P, Sachse M, Klumperman J, Strous GJ. Growth hormone receptor ubiquitination coincides with recruitment to clathrin-coated membrane domains. J Biol Chem. 2001; 276(6):3778-3784. (Clone-specific: Electron microscopy). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.