Integrin α2 Antibody (P1H5): sc-13546

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

Proteins from subconfluent (approximately 50–70%) cells were extracted in PBS containing 1% Triton X100 and protease, followed by incubation on ice for 30 min and centrifugation at 12,000 g for 1 minute. The protocol applied by Teittinen et al. [27] was followed to obtain nuclear extracts. Briefly, subconfluent cells were washed with PBS and collected by centrifugation. The cells were resuspended in 5 ml hypotonic buffer (10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT (Sigma)) and broken on ice using a Dounce homogenizer. Nuclei were pelletted by centrifugation (228 g, 5 min, +4°C) and purified by isopycnic centrifugation (1,430 g, 5 min, +4°C) on a two-step sucrose gradient: 250 mM sucrose, 10 mM MgCl2 vs 880 mM sucrose, 0.5 mM MgCl2. The proteins of this nuclear fraction were extracted as described above.
The protein concentration was determined by the Bradford method [28]. Then, 20 μg of cellular and 50 μg of nuclear protein were used for SDS-PAGE analysis according to Laemmli et al. [29] and transferred to Hybond-C extra nitrocellulose membrane (Amersham plc, Buckinghamshire, UK). Non-specific epitopes were masked, exposing membranes to 5% freeze-dried fat-free milk in TBS-T for 1 hour. Primary antibodies (see Table 1) were incubated for 2 hours. After washings, the blots were incubated with the secondary antibody: peroxidase-conjugated swine anti-rabbit and rabbit anti-mouse (DAKO, Clostrup, Denmark) 1:2,000, or Peroxidase Horse Anti-Goat IgG (H+L; Vector Laboratories) 1:10,000 for 1 hour. Subsequently, the blots were washed, and the signal was detected by enhanced chemiluminescent ECL reagent (Amersham) according to the manufacturer’s protocol. The blots were visualized on Super RX medical X-ray film (Fujifilm Corporation, Tokyo, Japan) and the bands quantitated by Kodak imaging software (Eastman Kodak Company, US). The protein expression was normalized to the house-keeping protein gamma-tubulin, and additionally to mitochondrial content (NAO result) in the case of the mitochondrial proteins.

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