The cells were lysed with immunoprecipitation assay buffer containing protease inhibitors. The protein concentrations were examined using the BCA Protein Assay kit (Vazyme Biotech, China).40 μg of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a piece of polyvinylidene diluoride membrane. The membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.5% of Triton X-100) for 1 hour at room temperature, and then incubated with the appropriate primary antibody against Col1a1 (No.ab21286 Abcam)and OPN (No. ab75285 Abcam) or GAPDH (No. 10494-1-AP Proteintech, China) overnight at 4°C. The membranes were washed 3 times in TBST, followed by incubation with the appropriate horseradish peroxidase-linked secondary antibodies (No. 10285-1-AP Proteintech, China) for 1 hour at room temperature. The specific proteins on the blots were developed with enhanced chemiluminescence (Vazyme Biotech, China) and visualized as the bands on the CL-XPosure Film (Thermo Fisher Scientific).
Ab75285
Manufactured by Abcam
Sourced in United Kingdom
Ab75285 is a laboratory reagent. It is a standardized and quality-controlled solution designed for use in various experimental protocols.
Automatically generated - may contain errors
Lab products found in correlation
Ab181602, by Abcam (2 mentions)
Enhanced chemiluminescence, by Vazyme (1 mentions)
Ab21286, by Abcam (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Vazyme (1 mentions)
No 10494 1 ap, by Proteintech (1 mentions)
No 10285 1 ap, by Proteintech (1 mentions)
Ab13420, by Abcam (1 mentions)
Ab8925, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Electrogenerated chemiluminescence, by Cytiva (1 mentions)
Ab23981, by Abcam (1 mentions)
Ab43791, by Abcam (1 mentions)
Ab93876, by Abcam (1 mentions)
Anti rabbit antibodies, by Abcam (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Ab51054, by Abcam (1 mentions)
Ab7800, by Abcam (1 mentions)
Ab5407, by Merck Group (1 mentions)
4 protocols using ab75285
1
Western Blot Protein Expression Analysis
2
Protein Expression Analysis of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted, and protein concentrations were quantified using the BCA Protein Assay Kit. The membrane was blocked and incubated with rabbit anti-Runx2 (1:1000, ab192256, Abcam), anti-OCN (1:1000, ab13420, Abcam), anti-OPN (1:1000, ab75285, Abcam) primary antibody, anti-NOTCH1 (1: 1000, ab8925, Abcam) and anti-GAPDH (1:10,000, ab181602, Abcam) at 4 °C overnight. Then a horseradish peroxidase-conjugated secondary antibody was added. Western blot analysis was performed according to the references (Welinder and Ekblad 2011 (link)). The western blot experiments were performed in three replicates.
3
Western Blot Analysis of Bone Marrow Stromal Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared from the BMSCs. Cells were lysed using mammalian protein extraction reagent (Pierce Chemical, Dallas, TX), containing protease inhibitor mixture (Roche Applied Science, Indianapolis, IN). The whole-cell lysates (10 μg protein/lane) were loaded and separated in 10% sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) gels, electro-blotted into a nitrocellulose membrane, and immune-blotted with anti-rabbit antibodies (Abcam, Cambridge, UK) to GAPDH (ab181602, 1:10,000), KLF2 (ab139699, 1:1000), runt-related transcription factor 2 (Runx2, ab23981, 1:1000), osteocalcin (OCN, ab93876, 1:500), osteopontin (OPN, ab75285, 1:1000), WWP1 (ab43791, 1:1000), CD63 (ab134045, 1:1000), CD81 (ab109201, 1:1000), Calnexin (ab92573, 1:20,000), TSG101 (ab125011, 1:1000), p65 (ab32536, 1:1000), phosphorylated p65 (ab86299, 1:2000), IκBα (ab32518, 1:1000), and phosphorylated IκBα (ab133462, 1:10,000). The electrogenerated chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) was employed to visualize these bands.
4
Localization of Opsin Proteins in Human Skin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Female human skin was obtained with full written consent adhering to the Declaration of Helsinki principles and under human tissue act guidelines. Facelift or abdominoplasty procedures were always performed in the morning and skin processed in the afternoon of the same day. Seven micrometer cryosections (n ¼ 4 donors, 44-63 years) were fixed in acetone before blocking with 5% bovine serum albumin (BSA) and 5% donkey serum (DKS) in phosphate-buffered saline (PBS) for 1 hour. Primary antibodies were diluted in 1% BSA and 1% DKS and incubated overnight at 48C; 1:200 OPN1-SW (AB5407, Millipore, Amsterdam-Zuidoost, the Netherlands), 1:100 OPN3 (ab66742 for immunohistochemistry (IHC) and ab75285 for immunocytochemistry (ICC), Abcam, Cambridge, UK), 1:200 OPN5 for IHC and 1:500 for ICC (ab199668, Abcam). A negative control (omission of primary antibody) was included in the experimental procedure. Double staining was performed with 1:200 KRT14 (ab51054 or ab7800, Abcam). Incubation with 1:200 Alexa-488 (ab150073, Abcam) and Alexa-647 (ab150115, Abcam) was for 1 hour at 378C. Slides were mounted using VECTASHIELD 1 containing DAPI (VECTOR). Images were taken using a confocal microscope (Leica Microsystems B.V., Amsterdam, the Netherlands).
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!