Total protein sample from BMSCs was isolated by RIPA lysis buffer and protease inhibitor, and sample was treated with electrophoresis for 70 min on 10% SDS-PAGE. After electroblotting to PVDF membranes (Millipore, Bellerica, MA), sample was incubated with 5% nonfat milk, prior to incubation with primary antibodies (Abcam, Cambridge, MA) against ALP (1:500, ab67228), RUNX2 (1:1000, ab23981), OSX (1:500, ab22552), OCN (1:500, ab93876), COL1A1 (1:1000, ab34710), RBPJ (1:1000, ab180588) and control (GAPDH, 1:1000, ab181602), as well as HRP-tagged secondary antibody (1:5000, ab175733). Protein levels were monitored by Image Quant LAS 4000 (GE Healthcare, Milwaukee, MI).
Ab67228
Manufactured by Abcam
Sourced in United Kingdom
Ab67228 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab93876, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab8448, by Abcam (2 mentions)
Ab209484, by Abcam (2 mentions)
Ab23981, by Abcam (1 mentions)
Ab180588, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab22552, by Abcam (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Ab214362, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab52128, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab6789 ab6721, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bca protein estimation kit, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
5 protocols using ab67228
1
Molecular Profiling of BMSC Osteogenesis
2
Osteogenic Protein Expression Analysis
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The hBMMSC lysates were extracted using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, USA), and protein quantification was performed using a BCA protein estimation kit (Thermo Fisher Scientific). Subsequently, the proteins were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Thermo Fisher Scientific). The membranes were blocked with BSA (5%; Sigma) dissolved in PBS containing 0.02% Tween 20 for 60 min. The membrane was initially incubated with primary antibodies at room temperature for 1 h, followed by incubation with secondary antibodies at 4°C overnight. The antibodies used are listed as follows: anti-OCN (ab93876, 1:2000; Abcam, Cambridge, UK), anti-OPN (ab8448 1:2000; Abcam), anti-RUNX2 (ab192256, 1:500, Abcam), anti-ALP (ab67228, 1:2000; Abcam), anti-BSP (ab52128, 1:2000; Abcam), anti-Bcl-2 (ab59348, 1:5000; Abcam), anti-p-ERK1/2 (ab214362, 1:500; Abcam), anti-ERK1/2 (ab54230, 1:1000; Abcam), anti-β-actin (ab8227, 1:5000; Abcam), and HRP-conjugated goat anti-mouse/rabbit (ab6789/ab6721, 1:5000; Abcam). Protein bands were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific), and gray images and values were recorded using a Blot Scanner (LiCor, Lincoln, USA).
3
Protein Expression Analysis of Osteoblast Markers
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The RIPA buffer containing protease inhibitors (Beyotime, Shanghai, China) was used to extract protein samples from target cells. The BCA quantitative method (Beyotime) was used to quantify the protein sample concentration. SDS-PAGE (10%; Invitrogen) was used to separate collected protein samples, which were then transferred to PVDF membranes (Millipore, Burlington, MA, USA). BSA (5%) was used to incubate the membranes for 2 h to prevent non-specific bindings. The following antibodies were used to incubate the membranes at 4°C overnight: ALP (ab67228, Abcam), OCN (ab93876, Abcam), RUNX2 (ab76956, Abcam), Osterix (ab209484, Abcam), DLX3 (ab178428, Abcam). Proper secondary antibodies (Cowin Biotech Co, Beijing, China) were used to incubate the membranes at room temperature for 2 h after the primary antibody incubation. Enhanced Chemiluminescence (ECL) Fluorescence Detection Kit (BB-3501; Amersham Pharmacia, Piscataway, NJ, USA) was used to visualize the blot signal on a Bio-Rad image analysis system (Bio-Rad, Hercules, CA, USA). The relative protein content is expressed by the gray value of the corresponding protein band/GAPDH protein band.
4
Protein Expression Analysis in Cell Lysates
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The total proteins were extracted from cells by RIPA lysis buffer (Beyotime) with phenylmethanesulfonyl fluoride (PMSF; Beyotime) and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime) transferring to polyvinylidene difluoride membranes (PVDF; Millipore, MA, USA). Primary antibodies against type I collagen (COL1) (1:1000, Bioss, Beijing, China), runt-related transcription factor-2 (RUNX2) (1:1000, 12556, CST, MA, USA), ALP (1:1000, ab67228, Abcam), NRF2 (1:1000, ab62352, Abcam), NAD(P)H quinone oxidoreductase 1 (NQO1) (1:10,000, ab80588, Abcam), CD206 (1:1000, ab125028, Abcam), iNOS (1:1000, 18985-1-AP, Proteintech, Wuhan, China), IDO (1:1000, ab211017, Abcam), IL-1β (1:1000, A1112, Abclonal), AhR (1:1000, ab190797, Abcam), PCNA (1:500, bs-0754R, Bioss), GAPDH (1:2000, Bioss) were used. The secondary antibody goat anti-rabbit IgG H&L (HRP) (1:5000, ab205718, Abcam) was used. The images were detected by using ChemiDocTM MP Imaging System (Bio-Rad, USA).
5
Western Blot Analysis of BMSC Differentiation
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After transfection for 7 days, BMSCs were collected and analyzed via Western blotting. The cells were lysed in radioimmunoprecipitation assay buffer containing proteinase inhibitors. Protein samples were separated on 10% gels by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were electroblotted onto polyvinylidene difluoride membranes (0.45 mm; Millipore, Billerica, MA, USA). Next, the membranes were blocked using 5% nonfat dry milk prepared in Tris-buffered saline containing Tween 20 for 1 h. The membranes were then probed overnight with the following primary antibodies: anti-Runx2 antibody (1:1000; #12556; Cell Signaling Technology, Boston, MA, USA), anti-OPN antibody (1:1000; ab8448; Abcam, Cambridge, UK), anti-Osterix antibody (1:1000; ab209484; Abcam), anti-ALP antibody (1:500; ab67228; Abcam), and anti-GAPDH antibody (1:2500; ab9485; Abcam) at 4°C. This was followed by incubation with an HRP-conjugated secondary antibody (1:2000; ab97051; Abcam) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence system, and the levels of the experimental proteins were normalized to those of actin. The levels of phosphorylated proteins were normalized to those of the corresponding total proteins.
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