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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Growth-factor Receptor-Bound Protein 2 (GRB2) was isolated by using the EGF receptor C-terminus as a probe in the screening of λgt11 expression libraries. This 24 kDa GRB2 protein is ubiquitously expressed and consists of one SH2 domain flanked by two SH3 domains. Its ability to bind proteins through these domains has prompted much investigation of its role as an adaptor protein. Sos, a Ras GDP/GTP exchange protein, is constitutively bound to the SH3 domain of GRB2. Following growth factor stimulation, receptor tyrosine kinases autophosphorylate, creating a binding site for the GRB2 SH2 domain. Alternatively, GRB2 interacts with the receptor-bound active Shc protein.Through these interactions, the GRB2/Sos complex is translocated to the membrane where Sos activates membrane-bound Ras to initiate the Ras signaling pathway. Other proteins that similarly interact with GRB2 include Cbl, PTP1D, and Dynamin. However,the mechanisms through which these associations impact cellular responses remain to be discovered.
Development References (5)
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Elowe S, Holland SJ, Kulkarni S, Pawson T. Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction. Mol Cell Biol. 2001; 21(21):7429-7441. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Lowenstein EJ, Daly RJ, Batzer AG, et al. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992; 70(3):431-442. (Biology). View Reference
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Miki H, Miura K, Matuoka K, et al. Association of Ash/Grb-2 with dynamin through the Src homology 3 domain. J Biol Chem. 1994; 269(8):5489-5492. (Biology). View Reference
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Saucier C, Papavasiliou V, Palazzo A, Naujokas MA, Kremer R, Park M. Use of signal specific receptor tyrosine kinase oncoproteins reveals that pathways downstream from Grb2 or Shc are sufficient for cell transformation and metastasis. Oncogene. 2002; 21(12):1800-1811. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Zeng L, Sachdev P, Yan L, et al. Vav3 mediates receptor protein tyrosine kinase signaling, regulates GTPase activity, modulates cell morphology, and induces cell transformation. Mol Cell Biol. 2000; 20(24):9212-9224. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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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.
