Total protein was extracted from cells and tissues with SDS lysis buffer (Beyotime), with the concentration measured utilizing a bicinchoninic acid kit (20201ES76, YEASEN Biotechnology Co., Ltd., Shanghai, China). After separation using 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the sample was submitted to electrotransfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) which were blocked using 5% blocking solution with skimmed milk powder and underwent overnight incubation at 4°C with primary rabbit antibodies against CGRP (ab189786, 1 : 1000, Abcam), RUNX2 (ab76956, 1 : 1000, Abcam), OCN (ab93876, 1 : 500, Abcam), ALP (ab229126, 1 : 1000, Abcam), CD45 (ab40763, 1 : 5000, Abcam), CD73 (ab133582, 1 : 5000, Abcam), and CD90 (ab92574, 1 : 1000, Abcam) as well as 1 h-incubation with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (ab150077, 1: 1000, Abcam) at ambient temperature. The immunocomplexes on the membrane were visualized utilizing enhanced chemiluminescence (ECL) reagent (ECL808-25, Biomiga, USA) at ambient temperature for 1 min, and band intensities were quantified with the help of Image Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD, USA).
Ab229126
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab229126 is a primary antibody that binds to a specific target. It is designed for use in various laboratory applications, such as immunoassays and immunohistochemistry. The core function of this product is to provide specific and reliable detection of its target.
Automatically generated - may contain errors
Lab products found in correlation
Ab236639, by Abcam (8 mentions)
Ab209484, by Abcam (6 mentions)
Ab8245, by Abcam (5 mentions)
Ab93876, by Abcam (4 mentions)
Ab23981, by Abcam (4 mentions)
Ab9485, by Abcam (4 mentions)
Ab133612, by Abcam (4 mentions)
Ab8448, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab236618, by Abcam (3 mentions)
Ab181602, by Abcam (3 mentions)
Easyblot ecl, by Sangon (3 mentions)
Total protein extraction kit, by Sangon (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ab76956, by Abcam (2 mentions)
Ab187647, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab189786, by Abcam (1 mentions)
Ab133582, by Abcam (1 mentions)
21 protocols using ab229126
1
Protein Expression Analysis of Stem Cell Markers
2
Osteogenic Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMSCs were lysed, and the protein concentration was measured by BCA method (Thermo Fisher Scientific) and adjusted by radioimmunoprecipitation (Beyotime, China). The proteins were separated by SDS-PAGE at 30 μg/lane concentration, and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After this, the membrane was soaked in 5% skimmed milk for 1 h at 25°C, and incubated overnight at 4°C with the antibodies against RUNX2 (ab23981, Abcam, UK), ALP (ab229126, Abcam), NNMT (ab119758, Abcam), and GAPDH (ab9485, Abcam). After washing with PBS, the membrane was incubated at 37°C for 1 h in appropriate secondary antibody. Finally, the relevant protein bands were detected using ECL kits (Santa Cruz, USA) and the expression profiles of different proteins were analyzed using Image J Software (Version 1.38; NIH, USA). GAPDH was used as an internal control.
3
Protein Expression Analysis of C2C12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2C12 cells were lysed with an ice-cold radio immunoprecipitation assay lysis buffer containing protease and phosphatase inhibitor cocktails (Abcam, Cambridge, UK). Protein lysates were cleared by centrifugation at 4 °C for 15 min, and the supernatants were collected for further experiments. 30 μg total protein samples (each lane) mixed with loading buffer (Beyotime) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). Primary antibodies used to detect specific proteins were: MT1/2 (Abcam, ab95042; 1:1000 dilution), MT3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-164990; 1:500 dilution), ALP (Abcam, ab229126; 1:1000 dilution), Runx2 (Abcam, ab236639; 1:1000 dilution), Osterix (Abcam, ab229258; 1:2000 dilution), Dlx5 (Abcam, ab109737; 1:3000 dilution), p-Smad1/5 (Cell Signaling Technology, Danvers, MA, USA, 9516; 1:1000 dilution), and GAPDH (Abcam, ab8245, 1:5000 dilution). After incubation with appropriate secondary antibodies, protein bands were visualized by using electrochemiluminescence reagent (Millipore), and images were captured by Amersham Image 600 system (GE Healthcare Life Sciences, Marlborough, MA, USA).
4
Cell Differentiation and Stimulation Assay Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture reagents, including fetal bovine serum (FBS; 10099141C), minimum essential medium α (α-MEM; C12571500BT), and penicillin/streptomycin (P/S; 15140122), were all purchased from Gibco (USA). Reagents for cell differentiation and stimulation included lipopolysaccharide (L8274; Sigma-Aldrich), cyclic polypeptide D7 (Scilight-Peptide Inc.), ascorbic acid (A8960; Sigma-Aldrich), β-glycerophosphate disodium (G9422; Sigma-Aldrich), dexamethasone (HY-14648; MCE), macrophage colony-stimulating factor (M-CSF; 416-ML-050; R&D Systems), and receptor activator of nuclear factor-κB ligand (RANKL; 462-TEC-010; R&D Systems). Primary antibodies that used in western blotting (WB) for the detection of ALP (ab229126), Runx2 (ab236639), and Ctsk (ab187647) were from Abcam; for Col1α1 (72026t) detection was from Cell Signaling Technology; for Nfatc1 (sc-7294) and Traf6 (sc-8409) detections were from Santa Cruz; for β-actin (hrp-66009) detection was from Proteintech. Secondary antibodies were purchased from Proteintech, including anti-rabbit (SA00001-2) and anti-mouse (SA00001-1).
5
Western Blot Analysis of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracted total protein from cultured cells with RIPA lysis buffer, and analyzed the protein content by BCA protein assay kit. Heated the protein at 95 °C for 5 min, and separated 20 μg protein on SDS-PAGE gels, next transferred them onto a PVDF membrane. The membranes were blocked with 5% skimmed milk powder for 1 h, then incubated together with the primary antibodies overnight at 4 °C. After washed the membranes with TBST buffer for 5 min three times, then incubated together with a corresponding secondary antibody for 1 h. After washing them with TBST three times, added ECL reagents to visualize the protein-antibody bound bands. Taking GAPDH as the internal reference, the relative protein expression was expressed by the ratio of the gray value of the target protein band to the gray value of the internal reference. The primary antibodies were as follows: anti-ITGA10 (AB6030, 1:1000, Merck), Alkaline phosphatase (ALP; ab229126, 1:1000, Abcam), Runx 2 (ab76956, 1:1000, Abcam), Osterix (ab209484, Abcam), Osteopontin (OPN; #88742, 1:1000, Cell Signaling Technology), p-PI3K (ab278545, 1:1000, Abcam), PI3K (#4249, 1:1000, Cell Signaling Technology), p-AKT (#4060, 1:2000, Cell Signaling Technology), AKT (#4691, 1:1000, Cell Signaling Technology), and GAPDH (ab8245, 1:5000, Abcam).
6
Western Blot Analysis of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular protein was isolated from the cells using the radio-immunoprecipitation assay (RIPA) buffer (Beyotime) containing protease inhibitors at 4 ºC for 30 min. Protein concentrations were measured with BCA protein, separated by SDS-PAGE (30 µg/well), and transferred to polyvinylidene fluoride membranes and blocked. The membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam). Afterward, these membranes were washed and followed by incubation with a secondary antibody (ab7090, Abcam). Eventually, an ECL kit (Millipore) was utilized to visualize the protein bands.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extraction was performed using cell lysis buffer (Ambion), and protein quality was determined using a bicinchoninic acid (BCA) protein assay (Pierce, USA). Briefly, 20 μg of protein was denatured at 95°C for 10 min in 5% β-mercaptoethanol solution and 95% Laemmli sample buffer (Bio-Rad). After separation by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Germany) and blocked for 1 h at 25°C using 0.5% skimmed milk. Thereafter, the membrane was incubated with ALP (ab229126; Abcam, USA), RUNX2 (ab236639; Abcam), and GAPDH (ab8245; Abcam) at 25°C for 1 h, followed by secondary antibodies (Bethyl, USA) at 25°C for 1 h. Membrane-bound antibodies were assessed by chemiluminescence after rinsing with PBS [32 (link)].
8
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were lysed in ice-cold RIPA buffer (Sabbiotech). Proteins were separated by SDS-PAGE, transferred to PVDF membranes and blocked with 5% milk for 1 h. Membranes were incubated with rabbit anti-BMAL1 (1:1000, #14020, CST), anti-beta actin (1:1000, #8457 S, CST), anti-Runx2 (1:1000, #ab236639, Abcam), anti-ALP (1:500, #ab229126, Abcam), anti-GADPH (1:1000, #ab8245, Abcam), anti-OSX (1:1000, #ab209484, Abcam), anti-YAP (1:1000, #1407 S, CST), anti-p-YAP (1:1000, #13008, CST), anti-MST1 (1:1000, #3682 S, CST), anti-p-MST1 (1:1000, #49332 S, CST), anti-LATS1/WATRS (1:1000, #ab243656, Abcam) 4 °C overnight, followed by a horseradish peroxidase-conjugated secondary antibody (1:5000, #L3012-2, Sabbiotech). Signals were visualized using an ECL detection kit (Thermo Fisher Scientific).
9
Osteogenic Differentiation Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteogenic medium, scaffolds, and seeded cells were collected and co-cultured for 21 days to obtain cells. RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was added to extract total cell proteins. Protein concentration was detected usingthe BAC method (Beyotime Biotechnology), and a 4 μg/μl protein solution was prepared. The proteinswereseparated by 12% SDS-PAGE electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, United States). After blockingwith 5% skimmed milk for 1 h, membranes were cultured with primary antibodies as follows: ALP (1:1000, ab229126, Abcam, Cambridge, United Kingdom), BSP (1:1000, #5468, Cell Signaling Technology), OCN (1:1000, ab133612, Abcam), OSX (1:1000, ab209484, Abcam), RUNX2 (1:1000, ab236639, Abcam), OCT4 (1:1000, ab181557, Abcam), SOX2 (1:1000, ab92494, Abcam), Nanog (1:1000, ab109250, Abcam), VEGF (1:1000, bs-0279R, Bioss), and GAPDH (1:2500, ab9485, Abcam). The next day, secondary antibodies (A0208, Beyotime Biotechnology) were added to the membrane, and the protein bands were detected usingECL (NCI5079, Thermo Fisher Scientific, Shanghai, China).
10
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
rotein contents were extracted from cells using radio immunoprecipitation lysis buffer (#89901; Thermo Fisher Scientific, Waltham, USA) containing proteinase and phosphatase inhibitors (Thermo Fisher Scientific), and the concentration was determined using a BCA protein assay kit (#P0011; Beyotime, Shanghai, China). After separated by SDS-PAGE, protein samples were transferred to the PVDF membrane (#GVWP02500; Sigma-Aldrich, St. Louis, USA). GAPDH was used as a loading reference. The membrane was subsequently incubated with primary antibodies against TGM2 (#ab2386; 1:1000, Abcam, Cambridge, UK), alkaline phosphatase (ALP; #ab229126; 1:500, Abcam), osteocalcin (OCN; #ab93876; 1:1000, Abcam), and runt-related transcription factor 2 (RUNX2; #ab236639; 1:1000, Abcam) at 4 °C overnight followed by further incubation with secondary antibodies. An enhanced chemiluminescence system (Kodak, Rochester, USA) was applied to visualize the signals on the membrane. ImageJ software (NIH, Bethesda, USA) was used analyze the signal intensity of the blots.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!