Product Name
ER beta antibody [14C8] (MS-ERB13-PX1)
Catalog Number
GTX70174
Product Description:
Mouse monoclonal antibody to human estrogen receptor beta
Manufacturer & Mfg Part#:
GeneTex GTX70174
Application Note:
For IHC, a recommended protocol is provided. WB: Use at a concentration of 1-5 µg/ml. Has only been tested in recombinant protein. Not tested in other applications. Optimal dilutions/concentrations should be determined by the researcher.
Background:
The human ER-beta is a newly discovered estrogen receptor initially cloned and characterized from testis. The size and structure of ER-beta is very similar to ER-alpha with the ligand and DNA binding domains being highly conserved, while the amino terminus which serves as their transactivation domain has diverged significantly. Similar in function to ER-alpha ER-beta binds to estrogen with a high affinity and regulates estrogen specific gene activation through direct interaction with estrogen response elements (ERE's).
Tested Applications:
Flow cytometry/FACS, Immunofluorescence, Immunohistochemistry, IHC (Formalin-fixed paraffin-embedded sections), Western blot. The usefulness of this product in other applications has not been determined.
Positive Control:
Recombinant fusion protein
Target Protein:
ER beta
Target Protein MW:
58-60kD
Clonality:
Monoclonal
Clone Number:
14C8
Purity:
Protein G Affinity Purified
Purification Note:
From ascitic fluid
Concentration:
1 mg/ml
Form:
Liquid
Storage Buffer:
Phosphate-bufferred saline, pH 7.4, containing no preservatives
Cross-Reactivity:
Human - Not yet tested in other species
Isotype:
IgG2b
Lightchain:
Kappa
Specificity:
Strong Erb staining in epithelial cell nuclei. Occasional weak to moderate staining is seen in surrounding stromal and endothelial cell nuclei. Sporadic light cytoplasmic staining is sometimes observed. We have also successfully detected ERb in colon and ovarian tumors using the same antibody.
Myeloma:
NS1
Immunogen:
Amino acids 1-153 of human ER-Beta expressed in E. coli.
Host:
Mouse
Storage:
Keep as concentrated solution. Store at 4ºC short term. For extended storage aliquot and store at -20ºC or below. Avoid freeze-thaw cycles.
General References:
1. Mosselman, S., Polman, J., and Dijkema, R. (1996) ERß: Identification and characterization of a novel human estrogen receptor. FEBS Letters 392:49-53.
1996
8769313
2. Dotzlaw, H., Leygue, E., Watson, P.H., and Murphy, L.C. (1996) Expression of estrogen receptor-beta in human breast tumors. J Clin Endo Metab 82:2371-2374.
1996
3. Kuiper, G.G.J.M., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J.A. (1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor ? and ß. Endocrinology 138:863-870.
1997
9048584
Specific References:
1. Speirs V, Skliris GP, Burdall SE, Carder PJ.. Distinct expression patterns of ER alpha and ER beta in normal human mammary gland.. J Clin Pathol. May;55(5):371-4.
2002
11986344
2. Shigehira Saji, Yoko Omoto, Chikako Shimizu, Margaret Warner, Yukiko Hayashi, Shin-ichiro Horiguchi, Toru Watanabe, Shin-ichi Hayashi, Jan-Åke Gustafsson, and Masakazu Toi . Expression of Estrogen Receptor (ER) ßcx Protein in ER{alpha}-positive Breast Cancer: Specific Correlation with Progesterone Receptor. Cancer Res., Sep; 62: 4849 - 4853.
2002
12208729
3. Suzanne A. W. Fuqua, Rachel Schiff, Irma Parra, John T. Moore, Syed K. Mohsin, C. Kent Osborne, Gary M. Clark, and D. Craig Allred. Estrogen Receptor ß Protein in Human Breast Cancer: Correlation with Clinical Tumor Parameters. Cancer Res., May; 63: 2434 - 2439.
2003
12750263
4. Konstantinopoulosa P.A et al.. Oestrogen receptor beta (ERb) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour¿s dedifferentiation. European Journal of Cancer 39:1251-1258.
2003
12763213
5. Skliris GP et al. Reduced expression of oestrogen receptor beta in invasive breast cancer and its re-expression using DNA methyl transferase inhibitors in a cell line model. J Pathol 201:213-20.
2003
14517838
6. Tee MK, Rogatsky I, Tzagarakis-Foster C, Cvoro A, An J, Christy RJ, Yamamoto KR, Leitman DC. Estradiol and Selective Estrogen Receptor Modulators Differentially Regulate Target Genes with Estrogen Receptors alpha and beta. Molec Biol Cell; 15:1262.
2004
14699072
7. Torsten A. Hopp, Heidi L. Weiss, Irma S. Parra, Yukun Cui, C. Kent Osborne and Suzanne A. W. Fuqua. Low Levels of Estrogen Receptor ß Protein Predict Resistance to Tamoxifen Therapy in Breast Cancer. Clinical Cancer Research Vol. 10, 7490-7499, November 15.
2004
15569979
8. Cooke F. J et al.. Epigenetic control of the oestrogen receptor genes in breast cancer.. British Journal of Surgery 90:42-43.
2003
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Submitted by: Marleen
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on 10/14/08
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Experiment performed: Immunohistochemistry Description:Dear Sir/Madam, we would like to perform IHC on parafinne-embedded tissues. Do you have a protocol for that? And what would you recommend as positive control?
Regards,
Marleen |
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GeneTex Response:
Protocol for detection of ERb in formalin-fixed paraffin-embedded human breast tissue using 14C8 monoclonal antibody
1. Cut 4-5 µ sections and mount onto Superfrost plus slides.
2. Dewax in xylene and rehydrate through graded alcohols.
3. Block with 3% H2O2 in methanol for 10 minutes.
4. Wash in running tap water for 5 minutes then block non-specific binding sites with normal horse serum.
5. To unmask the antigen, heat-mediated retrieval is required using either a microwave or pressure cooker. For microwave-mediated retrieval immerse sections in 1mM citrate buffer, pH 6, and microwaved at full power (600W) for 27 minutes (NB The exact time will depend on both the power and age of the microwave). Alternatively, sections can be pressure cooked for 4 minutes, immersed in 1mM citrate buffer, pH 6. Allow sections to cool for 20-30 minutes and wash in running tap water for 5 minutes.
7. Incubation sections with 5µg/ml primary antibody at 4°C overnight. Incubation time and temperature are critical.
8. Incubate with appropriate biotinylated secondary antibody for 30 minutes at room temperature, followed by streptavidin ABC kit (DAKO) according to the manufacturer's instructions.
9. Prepare 3,3¿-diaminobenzidine substrate and apply for 5-10 minutes.
10. Counterstain with haematoxylin, dehydrate and coverslip.
Using this protocol, we detect consistent, strong Erb staining in epithelial cell nuclei. Occasional weak to moderate staining is seen in surrounding stromal and endothelial cell nuclei. Sporadic light cytoplasmic staining is sometimes observed. We have also successfully detected ERb in colon and ovarian tumors using the same antibody.
Immunocytochemical analysis GTX70174 was conducted using Endocervical epithelium and Infiltrating lobular carcinoma of the breast.
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Submitted by: Yan
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on 10/14/08
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Experiment performed: not yet Description:Hi,
I tried to download your protocol for IHC using this antibody, but the link was blank. Could you please send me a protocol by email? Thanks a lot.
Yan |
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GeneTex Response:
Protocol for detection of ERb in formalin-fixed paraffin-embedded human breast tissue using 14C8 monoclonal antibody
1. Cut 4-5 µ sections and mount onto Superfrost plus slides.
2. Dewax in xylene and rehydrate through graded alcohols.
3. Block with 3% H2O2 in methanol for 10 minutes.
4. Wash in running tap water for 5 minutes then block non-specific binding sites with normal horse serum.
5. To unmask the antigen, heat-mediated retrieval is required using either a microwave or pressure cooker. For microwave-mediated retrieval immerse sections in 1mM citrate buffer, pH 6, and microwaved at full power (600W) for 27 minutes (NB The exact time will depend on both the power and age of the microwave). Alternatively, sections can be pressure cooked for 4 minutes, immersed in 1mM citrate buffer, pH 6. Allow sections to cool for 20-30 minutes and wash in running tap water for 5 minutes.
7. Incubation sections with 5µg/ml primary antibody at 4°C overnight. Incubation time and temperature are critical.
8. Incubate with appropriate biotinylated secondary antibody for 30 minutes at room temperature, followed by streptavidin ABC kit (DAKO) according to the manufacturer's instructions.
9. Prepare 3,3¿-diaminobenzidine substrate and apply for 5-10 minutes.
10. Counterstain with haematoxylin, dehydrate and coverslip.
Using this protocol, we detect consistent, strong Erb staining in epithelial cell nuclei. Occasional weak to moderate staining is seen in surrounding stromal and endothelial cell nuclei. Sporadic light cytoplasmic staining is sometimes observed. We have also successfully detected ERb in colon and ovarian tumors using the same antibody.
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Submitted by: Tina
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on 04/10/08
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I was checking the protocol for immunohistochemistry that you sent with the antibodies (GTX70174). It says that proposed concentration for primary antibdies is 5g/ml, but stock solution is 1mg/ml. This is probably mistake. Could you be so kind and tell me, what is the right/proposed dillution for IHC (DAB detection). |
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GeneTex Response:
We apologize for the confusion regarding the suggested primary antibody concentration. The correct primary antibody concentration we recommend for IHC-P is 5 µg/ml.
I have resent you a copy of the recommend IHC-P protocol for GTX70174 as an attachment.
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Submitted by: Elizabeth
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on 09/14/04
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Experiment performed: Western blot Description:Hello,
We were wondering what a recommended dilution is to perform a Western Blot, and could not find this information in the references listed. The stock solution is 1mg/ml. We are looking to use roughly 10mls of Ab mixture for our membrane.
Thank you,
The Luna Lab. |
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GeneTex Response:
Thank you for your inquiry. We recommend starting at a 1:500 dilution. Actual dilutions will vary depending on your sample. You may want to also refer to the following publication: Cancer Res., May 2003; 63: 2434 - 2439.
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