COL1A Antibody (COL-1): sc-59772

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:400) -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 (1:400) -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

Left side tissue samples of bilateral femora were acquired and homogenized using 100 µl ice-cold lysis buffer (10 mM EGTA, 20 mM Tris-HCl, 0.4% NaF, and protease inhibitors, 2 mM EDTA, pH 7.5) for 15 min. Subsequently, homogenates were centrifuged at 12,300 × g for 10 min at 4°C and a Bicinchoninic Acid protein assay (Beyotime Institute of Biotechnology, Nanjing, China) was used to determine the protein concentration. Next, 30–80 µg protein was separated using 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Blocking was performed with 5% skim milk powder in TBST for 1 h at 37°C. The following rabbit antibodies were used: PPARγ (1:300; sc-7273), AP2 (1:200; sc-12635), runt-related transcription factor (Runx)2 (1:400; sc-101145), Collagen (Col) I (1:400; sc-59772) and GAPDH (1:2,000; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. The membrane was extensively washed using TBST for 1 h and probed with horseradish peroxidase-conjugated secondary antibody (1:5,000; sc-2005 or sc-2768; Santa Cruz Biotechnology, Inc.) for 2 h. Relative band intensity was determined by a gel image analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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Manufacturer protocol

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