Preparation And Storage
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- 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.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
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Crk was first isolated as the v-crk oncogene from chicken retroviruses CT10 and ASV-1. All human cell lines examined to date express a 40 kDa Crk protein. In addition, there is variable expression of 42 kDa and 28 kDa Crk proteins. The c-crk gene is one of a class of genes, such as Nck and GRB2/ASH, which encode proteins that consist mainly of SH2 and SH3 domains. These proteins function as adaptor molecules in tyrosine kinase signal transduction pathways. The SH2 domains interact with phosphotyrosine-containing peptides, while the SH3 domains can enhance this interaction and/or bind to other cellular components. Both the SH2 and SH3 domains of the human Crk protein are required for differentiation of PC12 cells. Thus, Crk has a role in an NGF-induced signaling pathway that involves activation of p21ras. Furthermore, three proteins of 118 kDa, 125 kDa, and 136 kDa which specifically bind to the Crk SH3 domain have been identified.
Development References (7)
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Cho SY, Klemke RL. Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold. J Cell Biol. 2002; 156(4):725-736. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Fournier TM, Lamorte L, Maroun CR, et al. Cbl-transforming variants trigger a cascade of molecular alterations that lead to epithelial mesenchymal conversion. Mol Biol Cell. 2000; 11(10):3397-3410. (Clone-specific: Immunoprecipitation). View Reference
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Girardin SE, Yaniv M. A direct interaction between JNK1 and CrkII is critical for Rac1-induced JNK activation. EMBO J. 2001; 20(13):3437-3446. (Clone-specific: Immunofluorescence, Immunoprecipitation, Western blot). View Reference
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Liu J, Kimura A, Baumann CA, Saltiel AR. APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes. Mol Cell Biol. 2002; 22(11):3599-3609. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Miller CT, Chen G, Gharib TG, et al. Increased C-CRK proto-oncogene expression is associated with an aggressive phenotype in lung adenocarcinomas. Oncogene. 2003; 22(39):7950-7957. (Clone-specific).
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Smith JJ, Evans EK, Murakami M, et al. Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts. J Cell Biol. 2000; 151(7):1391-1400. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Smith JJ, Richardson DA, Kopf J, Yoshida M, Hollingsworth RE, Kornbluth S. Apoptotic regulation by the Crk adapter protein mediated by interactions with Wee1 and Crm1/exportin. Mol Cell Biol. 2002; 22(5):1412-1423. (Clone-specific).
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.