The extracted protein sample was separated using freshlyprepared sodium dodecyl sulphate polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene fluoride membranes, blocked with 5% BSA for 2 h, and probed with primary antibodies overnight at 4°C. The membranes were then re-probed with HRP-conjugated secondary antibody goat anti-rabbit (ab6721, 1:2,000; Abcam) at room temperature for 1 h. Next, enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA) was used to visualize the results by X-ray film. Protein bands were assayed utilizing Image J software, with β-actin selected for normalization. Primary antibodies used: SFRP2 (12189-1-AP, 1:1,000; Proteintech, Chicago, IL, USA), β-catenin (ab16051, 1:2,000, Abcam), p-β-catenin (ab75777, 1:500; Abcam), BAX (ab32503, 1:1,000; Abcam), Bcl-2 (ab196495, 1:1,000; Abcam), and β-actin rabbit polyclonal antibodies (ab8227, 1:2,000; Abcam).
Ab75777
Manufactured by Abcam
Sourced in United States
Ab75777 is a laboratory equipment product. It is a device used for a specific function in a laboratory setting. No further details are available.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Ab32391, by Abcam (2 mentions)
Ab75814, by Abcam (2 mentions)
Ab137852, by Abcam (2 mentions)
Ab252953, by Abcam (2 mentions)
Ab10485, by Abcam (2 mentions)
Ab190479, by Abcam (2 mentions)
Ab220030, by Abcam (2 mentions)
Ab115730, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab176316, by Abcam (2 mentions)
Ab151432, by Abcam (2 mentions)
Ab243861, by Abcam (2 mentions)
Ab282176, by Abcam (2 mentions)
Ab32072, by Abcam (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Ab16051, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
6 protocols using ab75777
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Vascular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from VSMCs or the aorta tissues according to the manufacturer’s instructions. In brief, equal amounts of protein was separated by SDS-PAGE and transferred onto an activated PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA) after electroblotting. After blocking with 5% non-fat milk for 2 h, the membrane was incubated with the primary antibodies overnight at 4 °C. The detailed information of primary antibodies were as follow: GADPH (ab181602, Abcam, 1:1000), phosphorylated-β-catenin (ab75777, Abcam, 1:500), β-catenin (ab32572, Abcam, 1:5000), Wnt inhibitory factor-1(WIF) (ab155101, Abcam, 1:2000), Runx2 (ab23981, Abcam, 1:1000), phosphorylated-AKT (ab81283, Abcam, 1:5000), Protein Kinase B (AKT) (ab188099, Abcam, 1:2000), receptor activator of NF-kB Ligand (RANKL) (ab239607, Abcam, 1:1000) and alkaline phosphatase (ALP) (ab228636, Abcam, 1:1000), phosphorylated-STAT3 (ab76315, Abcam, 1:2000), STAT3 (ab109085, Abcam, 1:1000), BMP2 (ab214821, Abcam, 1:1000). The membrane was rinsed with TBST three times (5 min/once), and then incubated at RT for 2 h with secondary antibody. After incubation, the membrane was rinsed three times with TBST (5 min/once). By using an enhanced chemiluminescence kit, protein bands were visualized by GeneGnome chemiluminescence imaging system.
3
Osteoblast Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After two washes with PBS, the cells were treated with radioimmunoprecipitation buffer (Beyotime) containing 1 mmol L−1 phenylmethyl sulfonyl fluoride. Following a 1 minute ultrasonic shock, the MC3T3-E1 cells were centrifuged at 12 000g and stored at 4 °C for 10 minutes. The cell fragments were then discarded to obtain the total protein. The proteins were loaded onto a 7.5% SDS-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was incubated in 5% bovine serum albumin for 2 hours and then with primary antibodies, including ALP (1 : 1000, Abcam, ab154100), RUNX2 (1 : 1000, Abclonal, A11753), β-catenin (1 : 1000, Abcam, ab265591), phosphorylation of β-catenin (1 : 1000, Abcam, ab75777), and GAPDH (1 : 2500, Abcam, ab9485), which were incubated overnight at 4 °C. The membrane was visualized using an enhanced chemiluminescent reagent (Abcam), and GAPDH was used as an internal control. Secondary antibodies (1 : 5000, ab6721, Abcam) were applied for 2 hours in the analysis.
4
Western Blot Analysis of Metabolic and Epigenetic Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples and cells were collected for Western blotting as previously described. Western blot analysis was performed using the following antibodies: LHPP (NBP1-83273, Novus, 0.2 ug/ml), METTL14 (ab220030, Abcam, 1:1000), HIF-1α (ab243861, Abcam, 1:1000), β-ACTIN (ab8226, Abcam, 1:2000), Acetyl Lysine (ab190479, Abcam, 1:1000), P300 (ab10485, Abcam, 1:5000), TIP60 (ab151432, Abcam, 1:1000), GCN5 (ab282176, Abcam, 1:1000), PCAF (ab176316, Abcam, 1:1000), GLUT1 (ab115730, Abcam, 1:5000), c-Myc (ab32072, Abcam, 1:1000), PKM2 (ab137852, Abcam, 1:1000), ALDOLASE (ab252953, Abcam, 1:1000), ENOLASE1 (H00002023-M01, Novus, 1:500), GLS1 (H00002744-M01, Novus, 1:500), GSK-3β (phospho S9) (ab75814, Abcam, 1:5000), GSK-3β (ab32391, Abcam, 1:5000), β-CATENIN (phospho S37) (ab75777, Abcam, 1:500).
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
The total protein content was isolated from the ligament tissues or cells by radio immunoprecipitation assay lysis buffer and centrifuged at 4˚C, 12,000 x g for 10 min. Protein concentration was determined using the bicinchoninic acid kit. The protein sample (30 µg) was separated by 8% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. A membrane blockade was conducted using PBS containing 5% skimmed milk for 2 h at room temperature. Subsequently, the primary antibodies were added for incubation at 4˚C overnight. The membranes were co-incubated with the HRP-conjugated goat anti-rabbit IgG (at a dilution ratio of 1:2,000, ab97051, Abcam) secondary antibody for 2 h. Protein bands were detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and estimated using the Image J software. Primary antibodies included in the present study were: runt-related gene 2 (RUNX2; at a dilution ratio of 1:5,000, ab76956, Abcam), Osteocalcin (at a dilution ratio of 1:1,000, ab133612, Abcam), DKK1 (at a dilution ratio of 1:1,000, ab109416, Abcam), Wnt1 (at a dilution ratio of 1:1,000, ab15251, Abcam), β-catenin (1:5,000, ab32572, Abcam), and p-β-catenin (at a dilution ratio of 1:5,000, ab75777, Abcam).
6
Western Blot Analysis of Metabolic Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples and cells were collected for Western blotting as previously described. Western blot analysis was performed using the following antibodies: LHPP (NBP1-83273, Novus, 0.2 ug/ml), METTL14 (ab220030, abcam, 1:1000), HIF-1α (ab243861, abcam, 1:1000), β-ACTIN (ab8226, abcam, 1:2000), Acetyl Lysine (ab190479, abcam, 1:1000), P300 (ab10485, abcam, 1:5000), TIP60 (ab151432, abcam, 1:1000), GCN5 (ab282176, abcam, 1:1000), PCAF (ab176316, abcam, 1:1000), GLUT1 (ab115730, abcam, 1:5000), c-Myc (ab32072, abcam, 1:1000), PKM2 (ab137852, abcam, 1:1000), ALDOLASE (ab252953, abcam, 1:1000), ENOLASE1 (H00002023-M01, Novus, 1:500), GLS1 (H00002744-M01, Novus, 1:500), GSK-3β (phospho S9) (ab75814, abcam, 1:5000), GSK-3β (ab32391, abcam, 1:5000), β-CATENIN (phospho S37) (ab75777, abcam, 1:500). *Adjuvant chemotherapy after surgery, no radiotherapy was administered to anyone of the patients enrolled.
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!