MA5-13304

antibody from Invitrogen Antibodies

Targeting: ESR1 ER-alpha, Era, ESR, NR3A1

Provider product page for MA5-13304

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [70]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [1]
Immunohistochemistry [1]
Other assay [6]

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Product number: MA5-13304 - Provider product page
Provider: Invitrogen Antibodies
Product name: Estrogen Receptor alpha Monoclonal Antibody (6F11)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA5-13304 has successfully been used in IHC, IP, and Western blot procedures. Paraffin sections require an antigen retrieval pretreatment with sodium citrate buffer (pH 6.0) prior to staining. Use Samboni’s fixative for optimal staining results. Positive control of Breast carcinoma suggested.
Reactivity: Human, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 6F11
Vial size: 500 µL
Concentration: Conc. Not Determined
Storage: 4° C

ESR1 protein structure - MA5-13304 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Estrogen Receptor alpha (ER alpha) was performed by loading the indicated amounts of recombinant full-length human ER alpha (Product # A15674) and ER beta (Product # A15664) proteins, and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane (Product # 88518) using the G2 Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. ER alpha was detected at ~66 kD (left panel) using an ER alpha monoclonal antibody (Product # MA5-13304) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:40,000. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). Directly alongside the above blot, a second blot containing the same recombinant ER alpha and ER beta protein samples was probed with an ER-beta specific polyclonal antibody (Product # PA1-313, right panel) at a dilution of 1:2000 overnight at 4C, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000. Chemiluminescent detection was again performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Estrogen Receptor alpha (ER alpha) was performed by loading 20 µg of whole cell lysates (WCL) or nuclear extracts (NE) from the indicated breast cancer cell lines, and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane (Product # 88518) using the G2 Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. ER alpha was detected at ~66 kD (top panel) using an ER alpha monoclonal antibody (Product # MA5-13304) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:40,000. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). To check for equivalent protein loading in each lane, a duplicate blot was probed with an HDAC1 polyclonal antibody (Product # PA1-860, bottom panel) at a dilution of 1:1000 overnight at 4C, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000. Chemiluminescent detection was also performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Estrogen Receptor using Estrogen Receptor Monoclonal Antibody (Product # MA5-13304) on MCF-7 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Estrogen Receptor using Estrogen Receptor Monoclonal Antibody (Product # MA5-13304) on denatured Human MCF-7 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Nf1 deficiency induces ER + /PR + mammary tumors. We observed mammary tumors with diverse mammary histology in G0 - G2 animals. a Nf1 females and b Nf1 males developed mammary adenocarcinomas with mixed histology including acinar, cystic, papillary, ductal, and solid features. All images were taken at 100X magnification. c Immunostaining of Nf1 mammary tumors ( rNf1 #6) for estrogen receptor, progesterone receptor, HER2, Ki67, and pan-cytokeratin (AE1/AE3). All immunostaining images were taken at 200X magnification

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1 ESR1 mutant models exhibited oestrogen-independent growth and metastatic propensity. a MTT growth assay of Tam and Ful-treated MCF-7 and b T47D cells. Percent survival is normalised to no treatment controls. Graphs represent mean + standard deviation ( N = 3 replicates) one-way ANOVA was used for statistical analysis. c MCF-7 cells were cultured in charcoal-stripped serum supplemented media, underwent whole cell lysis, and analysed by immunoblot for ER-regulated proteins (left). Values below PR, CCND1 and c-Jun represent normalised densitometry of each protein compared to GAPDH. PR was not detected in WT cells; thus, relative expression was normalised to Y537S cells. Cells were cultured in charcoal-stripped supplemented media and underwent cellular fractionation (right). Nuclear fraction is shown. d T47D cells were cultured in charcoal-stripped serum supplemented media and analysed by immunoblot for ER-regulated proteins (left). Values below PR, CCND1 and c-Jun represent normalised densitometry of each protein compared to GAPDH. PR was not detected in WT cells. Cells were cultured in charcoal-stripped supplemented media and underwent cellular fractionation (right). e Venn diagram of upregulated MSigDB Hallmark gene sets in MCF-7 ESR1 Y537S and LTED cells compared to MCF-7 ESR1 WT, and T47D ESR1 Y537S compared to T47D ESR1 WT. The numbers shown are of Hallmarks with a false discovery rate < 0.25. f In vivo individual tumour growth curves of MCF-7 ESR1 WT, ESR1 Y537S and

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 ESR1 mutations induced a global kinome reprogramming. a Flowchart demonstrating the total number of kinases captured in KiP experiment and the selection of kinases used to perform KEGG pathway analysis. Hyperactivated was defined as Y537S/WT >= 1.5-fold. Unique was defined as having corresponding peptide detection by MS in only MCF-7 ESR1 WT or MCF-7 ESR1 Y537S cells. b KEGG pathway analysis of the hyperactivated kinases in ESR1 Y537S cells. Analysis was performed using the DAVID Bioinformatics Functional Annotation Tool. c Y537S/WT quantitative ratio of kinases in KiP analysis categorised by RTKs, PI3K, and MAPK pathways. Red bars represent Y537S-unique activated kinases. d MCF-7 cells were cultured in charcoal-stripped serum-supplemented media and analysed by immunoblot for phosphorylated and total RON. Numbers below each protein represent normalised densitometry of each protein compared to GAPDH. e T47D cells were cultured in charcoal-stripped serum-supplemented media and analysed by immunoblot for phosphorylated and total RON. Numbers below each protein represent normalised densitometry of each protein compared to GAPDH. f MCF-7 cells were cultured in full serum-supplemented media and treated with BMS-777607/ASLAN002 (RONi) for 90 min and analysed by immunoblot for phosphorylated and total RON and IGF-1R. Numbers below each protein represent normalised densitometry of each protein compared to GAPDH. g MCF-7 cells were cultured in full serum-supplemented media and t

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 ET reduced RON/PI3K pathway activation. a Fold change of kinases in KiP analysis in MCF-7 Y537S cells after treatment with Tam. X -values represent fold change of Tam-treated Y537S cells compared to untreated Y537S cells. b MCF-7 cells were cultured in 10% FBS supplemented media and treated with 1 uM Tam and 1 uM Ful for 48 h. Immunoblot analysis of phosphorylated and total RON was performed. Numbers below each protein represent normalised densitometry of each protein compared to GAPDH. c MCF-7 cells were cultured in 10% FBS supplemented media and treated with Tam and Ful for 48 h. qRT-PCR analysis of RON was performed. Graphs represent mean + standard error of the mean ( N = 3 replicates). Two-way ANOVA was used for statistical analysis. d MCF-7 cells were cultured in 10% FBS supplemented media and treated with RONi for ninety minutes. Immunoblot analysis was performed for phosphorylated and total AKT and p44/42 MAPK. e MCF-7 cells were cultured in charcoal-stripped serum supplemented media and treated with RONi for 24 hours and ER transactivation assay was performed. Graphs represent mean + standard deviation ( N = 3 replicates). Student's t -test was used for statistical analysis. f MCF-7 cells were cultured in 10% FBS supplemented media and treated with 1 uM Tam and 1 uM Ful for 48 h. Immunoblot of phosphorylated and total AKT and p44/42 MAPK was performed. g T47D cells were cultured in 10% FBS supplemented media and treated with Tam and Ful for 48 h. Immunoblot of

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 MCF7-derived ducts grown in a mixed collagen:fibroblast matrix express lower levels of ER protein. MCF7-derived ducts were cultured alone or in co-culture with HMFs, treated with a vehicle control or 100 nM E2 for 24 hours. ( A ) Cultures were fixed and stained to visualize nuclei and ERalpha protein. Scale bar represents 100 mum. To quantify ER protein, the Hoescht stain was used to generate nuclear masks then ER expression was quantified within each nuclear mask. ( B ) Mean ER expression per condition was graphed to visualize ER protein changes within the entire population. A two-way ANOVA was run to evaluate interactions between conditions, followed by a Tukey's multiple comparison test to identify significant differences. ( C ) A density plot was created to show the frequency of individual cells (x-axis) that expressed various levels of ER protein (y-axis). *= vs. respective vehicle (p < 0.05) and ++= vs. respective monoculture (p < 0.05). Data is representative of three independent experiments.