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Purified Mouse Anti-SRC-1
Purified Mouse Anti-SRC-1

Western blot analysis of SRC-1 on a Jurkat lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the SRC-1 antibody.

Purified Mouse Anti-SRC-1

Immunofluorescent staining of A549 (ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the alcohol perm protocol and the anti-SRC-1 antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  Images were taken on a BD Pathway™ 855 Bioimager using a 20x objective.  This antibody also stained HeLa (ATCC CCL-2)and U-2 OS (ATCC HTB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Western blot analysis of SRC-1 on a Jurkat lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the SRC-1 antibody.

Immunofluorescent staining of A549 (ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the alcohol perm protocol and the anti-SRC-1 antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  Images were taken on a BD Pathway™ 855 Bioimager using a 20x objective.  This antibody also stained HeLa (ATCC CCL-2)and U-2 OS (ATCC HTB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat (Tested in Development)
Mouse IgG2a
Human SRC-1 aa. 761-863
Western blot (Routinely Tested), Bioimaging (Tested During Development)
165 kDa
250 µg/ml
AB_399738
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

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

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. 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.
  4. 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.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Triton is a trademark of the Dow Chemical Company.
612378 Rev. 3
Antibody Details
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8/SRC-1

Signal transduction via nuclear hormone receptors is important for cell growth and differentiation, development, and homeostasis. Nuclear hormone receptors are ligand-activated transcription factors that modulate target gene expression.These ligand/receptor complexes also interact with transcriptional coactivators which enhance ligand-dependent transcription. Various classes of coactivators have been identified, including SRC-1 and its related proteins, such as TIF-2/GRIP-1, RIP140, AIB-1, and TIF-1α and -1ß. SRC-1 is a member of the basic helix-loop-helix-PAS domain family. It contains an N-terminal basic-helix-loop-helix (bHLH), two N-terminal PAS domains, and a glutamine-rich region near the C-terminus. SRC-1 interacts with many different steroid receptors and recruitment of SRC-1 to promoter sites may facilitate opening of chromatin structure via its histone acetyltransferase activity, as well as stabilization of the preinitiation complex. ERK-2 can phosphorylate SRC-1 at threonine and serine sites in vitro. In addition, SRC-1 expression in cells stimulated with EGF enhances progesterone receptor-mediated activation of a target reporter gene. Thus, SRC-1 is an important transcriptional co-activator involved in steroid receptor signaling.

612378 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
612378 Rev.3
Citations & References
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Development References (4)

  1. Onate SA, Tsai SY, Tsai MJ, O'Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science. 1995; 270(5240):1354-1357. (Biology). View Reference
  2. Rowan BG, Weigel NL, O'Malley BW. Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway. J Biol Chem. 2000; 275(6):4475-4483. (Biology). View Reference
  3. Spencer TE, Jenster G, Burcin MM, et al. Steroid receptor coactivator-1 is a histone acetyltransferase. Nature. 1997; 389(6647):194-198. (Biology). View Reference
  4. Yao TP, Ku G, Zhou N, Scully R, Livingston DM. The nuclear hormone receptor coactivator SRC-1 is a specific target of p300. Proc Natl Acad Sci U S A. 1996; 93(20):10626-10631. (Biology). View Reference
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612378 Rev. 3

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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.