After 16 days, osteogenic media were removed before PBS washing and scraping of cells and mineralized nodules in cold RIPA buffer supplemented with a final concentration of 2 % SDS, 1 % deoxycholate sodium and 2× protease inhibitors (Pierce). Total proteins were extracted at 4 °C using 30 min of vortexing and sonication. Following protein concentration determination as stated in section 2.2 , 20 μg of proteins per sample were labeled with Cy5 dye (Amersham QuickStain Protein Labeling kit) according to the manufacturer's instructions and separated using 8 % SDS–PAGE. Following blotting on nitrocellulose in methanol, Cy5-labeled proteins were detected using the Amersham Imager 600 (GE Healthcare). After blocking in TBST with 5 % nonfat dry milk, the membrane was incubated overnight at 4 °C with a monoclonal rabbit antibody against type I collagen (Ab138492, diluted 1:1000, Abcam) in TBST with 1 % milk. Following washing steps and incubation with the secondary anti-rabbit HRP antibody (at 1/15000th, STAR124P, BioRad), imaging of type I collagen protein (COLI) was acquired using ECL Prime (Amersham). The signal intensities of Cy-5-labeled proteins and COLI were measured using Image Quant TL analysis software (Amersham) to express the quantity of COLI according to the amount of total loaded and blotted proteins (i.e., sum of the intensities of Cy-5-labeled proteins).
Star124p
Manufactured by Bio-Rad
Sourced in United States
The STAR124P is a lab equipment product manufactured by Bio-Rad. It is designed to perform specific laboratory functions, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Ab138492, by Abcam (1 mentions)
Image quant tl analysis software, by Cytiva (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Las4000 system, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Thrombin, by Thermo Fisher Scientific (1 mentions)
Star207p, by Bio-Rad (1 mentions)
Glutathione resin, by GenScript (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
4 protocols using star124p
1
Quantifying Type I Collagen in Osteogenic Cultures
2
ChIP-Western Assay for SATB2 Validation
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Efficiency of ChIP for mouse SATB2 and validation of mammalian SATB2 antibody was performed by ChIP western assay. Briefly, ChIP was performed with in house anti-human Satb2 antibodies. After bead washing and removal of excess TE buffer, beads were resuspended in 1× PBS and treated with 2 µl of DNAse (10 mg/ml) for 30 min at 37 °C. Further, beads were washed with 1× PBS twice and boiled in 2× Laemmli buffer (0.25 M Tris-Cl pH 6.8, 1% SDS, 1% β-mercaptoethanol, 15% glycerol) at 98 °C for 5 min and electrophoresed on 7.5% SDS-PAGE gel. Proteins were transferred to a 0.45 µM PVDF membrane (Millipore, USA) by wet transfer method at 0.6 A for 2.5 h at 4 °C. Non-specific sites on the membrane were blocked using 5% BSA and further incubated with anti-SATB2 antibody (1:1000 abcam ab34735) in 0.3% BSA overnight at 4 °C with constant rocking. Next day, the membrane was washed with 1× TBST (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% Tween-20) four times for 7 min each and incubated with anti-rabbit HRP conjugated antibody (1:10,000 STAR124P, Bio Rad, USA) in 1X TBST for 45 min at RT. After removing excess secondary antibodies by repeated washes with 1× TBST, signal was developed with Clarity western ECL substrate (Biorad) and captured using the LAS 4000 system (GE Healthcare, USA).
3
Recombinant Protein Purification and Antibody Generation
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The N-001 was prepared as described previously24 (link),25 (link). In brief, plasmid encoded constructs for C2I and C2II-C1 were expressed in E. coli BL21. E. coli was cultured in LB medium supplemented with ampicillin (100 µg/mL) at 37 °C and were induced at an optical density of ~ 0.6 at 600 nm wavelength with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). A French pressure cell was used to lyse the cell paste at 690 bar. Glutathione resin (Genscript) was used for affinity purification. GST fusion tags were removed using thrombin (Thermo Fisher). C2II-C1 was further activated using trypsin by incubation at 37 °C for 30 min at a 1:5 enzyme to substrate ratio as previously described45 (link). A custom-made polyclonal antibody targeting the N-001 enzymatic component C2I, was prepared in rabbits. The other antibodies used were goat anti-mouse IgG (H/L) polyclonal antibody (BioRad, Cat no. STAR207P) and goat anti-rabbit IgG (H/L): HRP (BioRad, Cat no. STAR124P). Thermo Scientific Pierce TMB substrate was used for ELISAs.
4
Immunoblot Analysis of Acetylated Proteins
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HeLa cells were lysed by boiling in 2% lithium dodecyl sulfate (LDS) (1x NuPAGE LDS Sample Buffer, ThermoFischer Scientific), followed by sonication to disrupt genomic DNA. Proteins were separated on a 4-12 NuPAGE gel (ThermoFischer Scientific) and transferred to a nitrocellulose membrane (BioRad). Acetylated proteins were visualized by immunoblot using pan-anti-acetylated lysine antibody (Immunechem #ICP0380) at a 1/1000 dilution and goat anti-rabbit horseradisch peroxidase (HRP) (BioRad, STAR124P) secondary antibody at a 1/5000 dilution.
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