Total proteins were isolated using RIPA lysis buffer with added PMSF and phosphatase inhibitors (BEYOTIME), and were quantified using a BCA protein assay kit (BEYOTIME). The proteins were separated using 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After being blocked with 5% fat-free milk, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-Periostin (Abcam, ab14041, 1:1000), mouse anti-β-actin (BOSTER, BM0627, 1:200), rabbit anti-ILK (Abcam, ab76468, 1:1,000), rabbit anti-pAkt (CST, 4060, 1:2,000), rabbit anti-Akt (CST, 4691, 1:1,000), rabbit anti-pGSK3β (Abcam, ab68476, 1:1,000), rabbit anti-GSK3β (Proteintech Group, Inc., 22104-1-AP, 1:1,000). Then, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (BOSTER, BA1051, 1:50,000) or HRP-conjugated goat anti-rabbit IgG (BOSTER, BA1054, 1:50,000) for 2 h at 37°C. The blots were visualized with Pierce™ ECL western blotting substrate (Thermo, NCI5079) and then exposed to X-ray films (Kodak, XBT-1). The relative protein levels were quantified using Image J software (NIH, Bethesda, MD, USA).
Bm0627
Manufactured by Abcam
Sourced in China
BM0627 is a lab equipment product. It is a specific tool used for scientific research and analysis purposes. The core function of this product is to perform a specific task within a laboratory setting. No further details can be provided about its intended use or features without the risk of introducing bias or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ba1054, by Boster Bio (2 mentions)
Pmsf and phosphatase inhibitor, by Beyotime (1 mentions)
Ba1051, by Boster Bio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Xbt 1, by Kodak (1 mentions)
Ab92324, by Abcam (1 mentions)
Cell lysate, by Beyotime (1 mentions)
Ab227560, by Abcam (1 mentions)
Ab187647, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab9957, by Abcam (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Ab131109, by Abcam (1 mentions)
Ab131485, by Abcam (1 mentions)
Ba1050, by Boster Bio (1 mentions)
5 protocols using bm0627
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of Osteoclast Markers
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Total protein was prepared using cell lysates (Beyotime, China), loaded on 10% or 12% SDS‐PAGE gels and then transferred to polyvinylidene fluoride membranes (Millipore, Germany). Membranes were blocked with 5% non‐fat milk at room temperature for 2 hours and incubated with primary antibodies against GAPDH (1:1000, Proteintech # 10494‐1‐AP), β‐actin (1:1000, Boster # BM0627), A20 (1:1000, Abcam # ab92324), RANKL (1:1000, Abcam # ab9957), OPG (1:1000, Abcam # ab73400), HIF‐1α (1:1000, CST # 36169), LC3B (1:2000, Abcam # ab51520), Beclin1 (1:1000, Proteintech # 11306‐1‐AP), p62 (1:1000, Proteintech # 18420‐1‐AP), ATG5 (1:1000, CST # 12994), TRAF6 (1:1000, Abcam # ab227560), CTSK (1:1000, Abcam # ab187647), TRAP (1:1000, Abcam # ab191406), MMP9 (1:500, Proteintech # 10375‐2‐AP), V‐ATPase D2 (1:1000, Invitrogen # PA5‐44359) overnight at 4°C. These blots were then incubated with secondary antibodies (1:8000, Proteintech # SA00001‐2) or (1:8000, Proteintech # SA00001‐1) for 50 minutes, and the band intensities determined by a chemiluminescent gel imaging system (Tanon, China).
3
Curcumin Effects on Endothelial Cells
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Cell suspension density was adjusted to (4–2) × 104/mL and then grown in chamber slides (Thermo Fisher Scientific, Shanghai, China). After static adherence, HemECs were exposed to 0 and 25 μM curcumin for 24 hours. Then cells were washed in phosphate buffer solution (PBS) for 3 times and fixed with 4% paraformaldehyde for 10 minutes. Permeabilization was performed by treatment with 0.1% Triton X-100 for 5 minutes. Endogenous catalase was neutralized by 0.3% H2O2. The cells were incubated with 10% goat serum in PBS to prevent nonspecific reaction and incubated with primary antibodies (β-actin, BOSTER BM0627, mouse polyclonal, 1:100; HIF-1α, Abcam 23426, rabbit polyclonal, 1:75; VEGF, BBI AB60788a, rabbit polyclonal, 1:300; Ang-2, BBI, AB20217a, rabbit polyclonal, 1:500) at 4°C overnight. After washes for 3 times, the cells were incubated with secondary antibody labeled fluorescence for 30 minutes, follow by 4’,6-diamidino-2-phenylindole (DAPI) staining. The cells were then examined and photographed using a fluorescence microscope (ZEISS, Germany).
4
Western Blot Analysis of Pancreatic and PSC Proteins
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For Western blotting, lysates from pancreatic tissue (loading protein = 40 µg) or PSCs (loading protein = 20 µg) were separated through sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with the respective primary antibody overnight at 4°C and then combined with secondary antibodies conjugated to horseradish peroxidase (BA1054 and BA1050; Boster Biological Technology Co., Ltd.). The primary antibodies used were anti‐p65 (ab131485; Abcam Inc), anti‐phosphorylated p65 (ab131109; Abcam Inc), anti‐α‐SMA (bs10196R; Beijing Biosynthesis Biotechnology Co., Ltd), anti‐IκB‐α (D120138; Sangon Biotech Co., Ltd) or anti‐β‐actin (BM0627; Abcam Inc). Detection and image capturing were performed using an enhanced chemiluminescence illuminating system.
5
Osteogenic Differentiation Markers of MC3T3-E1 Cells on Titanium Alloy Surfaces
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The four materials (cp-Ti, Ti-SLA, Ti-NW and Ti-NW-Zn) samples were prepared and applied into 6-well plates. Subsequently, MC3T3-E1 cells (2 × 105 cells/well) were seeded onto each surface of alloy and cultured for 7 days. Protein samples were extracted from cells by RIPA buffer after cell washing with pre-cooled PBS. After the electrophoresis, protein samples were transferred to a polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at room temperature (5% nonfat dry milk, in Tris-buffered saline containing 0.1% Tween-20; TBST). They were subjected to incubation with primary antibodies against Runx2 (12556; CST, Beverly, MA, USA), OSX (ab22552; Abcam, Cambridge, MA, USA) or GAPDH (BM0627, Boster, China) overnight at 4 °C. After TBST wash for three times, membranes were incubated with secondary antibodies (ZB-2301; Goat anti-Rabbit IgG, ZSGB-BIO, China) for 2 h, followed by chemiluminescence exposure using ECL Western Blot Kit (Millipore, USA). The background was subtracted, and the signal of each target band was normalized to that of the GAPDH band. Protein level was quantified using the Image J software k1.45 (version k1.45; National Institutes of Health, Bethesda, MA, USA). The experiment was performed in triplicate.
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