Recombinant Anti-Estrogen Receptor alpha (phospho S118) antibody [E91] (ab32396)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Posttreatment, cells were washed with phosphate-buffered saline (PBS) and lysed in-well with 2× SDS sample buffer (120 mM Tris-HCl, 20% glycerol, 2% SDS, 2% β-mercaptoethanol, 0.01% bromophenol blue, pH 6.8), followed by boiling at 95°C for 10 min. Protein concentrations were quantified using an RC DC kit (Bio-Rad, Hercules, CA), and equal levels of protein were run on a 10% SDS-PAGE gel. Blots were transferred to a polyvinylidene difluoride (PVDF) membrane, followed by blocking with 5% milk in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20). Primary antibodies to ER (1:1,000; HC-20, sc-543; Santa Cruz Biotechnology), pS118-ER #1 (1:1,000; #2511; Cell Signaling Technology), pS118-ER #2 (1:1,000; ab32396; Abcam), pS118-ER #3 (1:1,000; sc-12915; Santa Cruz Biotechnology), pS104/S106-ER (1:1,000; #2517; Cell Signaling Technology), pS167-ER (1:1,000; #5587; Cell Signaling Technology), pS294-ER (1:1,000; ab207602; Abcam), or β-actin (1:5,000; AC-15; Sigma) were diluted in 5% milk in TBST and incubated with blots overnight at 4°C. Blots were washed 3 times for 20 min each time in TBST, followed by 1 h of incubation with either sheep anti-mouse IgG–horseradish peroxidase (HRP; 1:3,000; NA931V; GE Healthcare) or donkey anti-rabbit IgG–HRP (1:3,000; NA934V; GE Healthcare) diluted in 5% milk in TBST. Blots were washed 3 times for 20 min each time in TBST, followed by one 20-min wash in TBS. Blots were then exposed to Clarity Western ECL solution (Bio-Rad), followed by exposure to autoradiography film (HyBlot CL; Denville Scientific).

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Estrogen Receptor alpha (phospho S118) antibody [E91] (ab32396) below.

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