Progerin Antibody (13A4D4): sc-81611

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:200) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:200) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Whole-cell lysates of MSCs were collected, proteins were extracted in NP40 cell lysis buffer (Invitrogen) with a protease and phosphatase inhibitor cocktail (ThermoScientific). Lysates were sonicated 4 times for 15 sec each, each with an interval of 15 sec in between, and then centrifuged at 10,000 g for 10 minutes at 4 °C. Protein concentration was measured using the Pierce BCA Protein Assay Kit (ThermoScientific) and the absorbance at 562 nm was evaluated using a Clariostar (BMG Labtech). A total of 20 μg of protein was loaded and run on a 7% tris-acetate gel (Criterion™ XT) using XT tricine running buffer (Biorad). Gels were then transferred onto polyvinylidene fluoride (PVDF) membranes (Biorad) using a Trans-Blot Turbo Transfer System (Biorad). Blots were blocked in Rockland blocking buffer (Millipore) diluted 1:2 in TBS 1x for 1 hour at room temperature. Membranes were incubated with primary antibodies diluted in blocking buffer with 0.1% Tween20 (VWR) overnight at 4 °C. The primary antibodies used here are a mouse anti-lamin A/C 1:200 (Millipore, JOL2, MAB3211), a mouse anti-progerin 1:200 (SantaCruz, 13A4D4, sc-81611) and a mouse anti-actin 1/5,000 (Millipore, MAB1501R). Washing was carried out for 45 minutes at room temperature with TBS + 0.1% Tween20 and the membranes were incubated with an IR-Dye 800CW conjugated with a secondary donkey anti-mouse antibody or a IR-Dye 680RD conjugated with a secondary donkey anti-mouse antibody (LI-COR Biosciences) at 1:10,000 in blocking buffer with 0.1% Tween20 and 0.01% SDS (Ambion). The detector was an Odyssey Infrared Imaging System (LI-COR Biosciences).

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Manufacturer protocol

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