Total protein from HCC tissues, matched normal adjacent tissue and cell lines (1×106 cells/well) was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein was quantified using a Bradford protein assay (Bio-Rad Laboratories, Inc.) and a Nanodrop spectrophotometer. An equal amount (25 µg) of protein was added to each well of 10% gels and resolved via SDS-PAGE. Proteins were transferred to PVDF membranes and were blocked by incubation for 1 h at 37°C with 5% non-fat powdered milk. The proteins on PVDF membranes were incubated with the following primary antibodies: PTTG1 (1:1,000; product code ab26273), N-cadherin (1:5,000; product code ab76011), vimentin (1:2,000; product code ab92547), Ki-67 (1:5,000; product code ab16667), E-cadherin (1:10,000; product code ab1416) and GAPDH (1:5,000; product code ab8245; all from Abcam) overnight at 4°C. Then, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (1:5,000; cat. no. ab6721; Abcam and 1:5,000; cat. no. ab205719; Abcam) at room temperature for 1 h. Finally, immunoreactive protein bands were visualized using an enhanced chemiluminescence solution (MilliporeSigma) and a ChemiDoc Imaging system (Bio-Rad Laboratories, Inc.). Protein expression was semi-quantified using Quantity One version 4.6 software (Bio-Rad Laboratories, Inc.).
Pttg1
Manufactured by Abcam
Sourced in United States
PTTG1 is a protein-coding gene that is involved in the regulation of cell cycle progression. It plays a crucial role in the control of sister chromatid separation during mitosis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Bio-Rad (1 mentions)
Quantityone version 4.6 software, by Bio-Rad (1 mentions)
Vimentin, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase linked secondary antibody, by GE Healthcare (1 mentions)
Foxm1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Notch2, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
3 protocols using pttg1
1
Western Blot Analysis of Protein Expression in HCC
2
Western Blot Analysis of Protein Expression
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Total cell lysate was prepared in RIPA buffer (Sigma, St. Louis, MO, USA) containing Protease Inhibitor Cocktail (Sigma). Protein concentrations were measured by Coomassie Plus Assay Kit (Pierce, Rockford, IL USA) using BSA as standard. Equal amounts (100 μg) of proteins were separated by NuPAGE Novex Bis-Tris Gels (Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated in TBS buffer containing 5 % nonfat dry milk (Bio-Rad, Hercules, CA, USA) for 1 hour at room temperature, followed by incubation with the primary antibodies at 4 °C overnight: FoxM1 (1:200), PTTG1 (1:500) and DKK1 (1:200) were from Abcam (Cambridge, MA, USA). β-actin (1:5000) was from Sigma. After washes with TTBS (0.5 % Tween-20 in TBS), membranes were subsequently incubated with horseradish peroxidase linked secondary antibody (GE Healthcare, Piscataway, NJ, USA) for 1 hour at room temperature and developed using ECL Western Blotting Detection Reagents (GE Healthcare). Detected bands were quantified using Image J v1.43 as instructed in the software manual.
3
Immunofluorescence and smFISH Analysis of Mouse Dental Tissues
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P1 and P3 mouse heads were embedded in paraffin, sectioned, and subjected to immunofluorescence, as described previously (Chiba et al., 2019 (link)). The primary antibodies of AMEL (Abcam, 1:200), NOTCH2 (Cell Signaling Technology, 1:200), PTTG1 (Abcam, 1:100), CLDN10 (Thermo Fisher Scientific, 1:200), ATF3 (Abcam, 1:100), and KRT15 (Abcam, 1:200) were used for immunostaining. These primary antibodies were detected by Alexa Fluor 488-conjugated antibody (Invitrogen, 1:400). Nuclear staining was performed with DAPI (Sigma).
For single-molecule fluorescence in situ hybridization (smFISH), Custom Stellaris® Probe Sets (LGC Biosearch Technology, Hoddesdon, United Kingdom) for mouse Ambn and mouse Dspp were designed by Stellaris Probe Designer. Hybridization buffer was prepared as previously described (Wang, 2019 (link)), and smFISH was performed following the manufacturer’s protocol.
For single-molecule fluorescence in situ hybridization (smFISH), Custom Stellaris® Probe Sets (LGC Biosearch Technology, Hoddesdon, United Kingdom) for mouse Ambn and mouse Dspp were designed by Stellaris Probe Designer. Hybridization buffer was prepared as previously described (Wang, 2019 (link)), and smFISH was performed following the manufacturer’s protocol.
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