For protein expression analyses, PC12 cells cultured in 60 mm petri-dishes (Sarstedt, Nürnbrecht, Germany) were lysed in 100 μl of RIPA buffer supplemented with 1 mM PMSF (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL, USA). Protein lysates (20 μg) were heated for 5 min at 94 °C in Laemmli sample buffer containing 5% β-mercaptoethanol and then loaded on 4–15% Tris-glycine SDS-PAGE gels, then transferred electrophoretically onto polyvinylidene difluoride (PVDF; Bio-Rad, Hercules, CA, USA) membranes at 200 mAmp for 2 h. Membranes were blocked with 5% non-fat dry milk for 1 h and incubated overnight at 4 °C with the antibodies specific to Shootin1, ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Thr308) and GAPDH (CST, Danvers, MA, USA). Protein bands were detected with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (CST; Danvers, MA, USA) and visualized by West-Femto ECL reagents (Pierce; Rockford, IL, USA). Chemiluminescent signals of immunoblots were documented using Gel Logic 2200 Pro (Carestream Health; Rochester, NY, USA). The net intensity of specific proteins was quantified using ImageJ (NIH, Bethesda, MD, USA).
Gel logic 2200 pro
Manufactured by Carestream
Sourced in United States
The Gel Logic 2200 Pro is a gel imaging system designed for capturing and analyzing gel electrophoresis images. It features a high-resolution camera and advanced optics for capturing detailed images of various types of gels, including agarose and polyacrylamide gels. The system provides users with the necessary tools for visualizing and quantifying nucleic acid and protein samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride pvdf, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions)
Biovision ecl western blotting substrate kit, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Coomassie blue g 250, by Merck Group (1 mentions)
Polyvinylpyrrolidone pvp, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
4 protocols using gel logic 2200 pro
1
Quantifying Protein Expression in PC12 Cells
2
Memantine's Effects on 4T1 Cell Signaling
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After 6 h, 24 h and 48 h treatment of 4T1 cells with memantine, cells were washed with PBS and scraped into the RIPA lysis buffer containing 1mM PMSF followed by sonication for 15 seconds. Samples were centrifuged for 15 minutes at 14000 rpm at 4 °C and the supernatant was collected. Proteins were quantifi ed by using the BCA Assay Kit (Thermo Pierce, Rockford, IL, USA). Protein lysates (20 μg) were heated for 5 minutes at 95 °C in LDS non-reducing sample buffer (Pierce, Rockford, IL, USA) and then loaded into the 10 % Tris-glycin gels. The gels were transferred to the PVDF membrane (Merck Millipore, Darmtadt, Germany) at 250 mAmp for 90 minutes. Membranes were blocked with 5 % non-fat milk powder in TBS-T for 1 hour at room temperature and incubated overnight at 4 °C with the primary antibodies for Bax, Bcl-2, Casp-3, Casp-9, E-Cad, Vimentin, B-Cat, GSK3B, p-ERK, ERK, p-GS, GS and Gapdh at 1:1000 dilution (Thermo Pierce, Rockford, IL, USA). Blots were washed with TBS-T subsequently. Protein bands were detected by using the secondary antibody (Thermo Pierce, Rockford, IL, USA) and the blots were visualized by BioVision ECL Western Blotting Substrate Kit (Biovision, California, USA). Chemiluminescent signals of immunob-lots were detected by using the Gel Logic 2200 Pro (Carestream Health, Rochester, NY, USA).
3
Protein Visualization via Coomassie and Silver
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SDS-PAGE gels were stained with Coomassie blue G-250 (Sigma, St. Louis, MO, USA), then with silver staining and visualized with GelLogic 2200Pro (Carestream, Rochester, NY, USA).
4
Western Blotting of Complement C9 Protein
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SDS-PAGE electrophoresis was performed on C9 (Sigma, St. Louis, MO, USA, Product No. C3660). Then, proteins in SDS-PAGE gel were transferred onto polyvinylidene difluoride (PVDF) (Themo Scientific, Waltham, MA, USA) blotting membrane. WB was run in an ice-cooled transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) filled tank at 150 V for an hour. The membrane was then incubated in polyvinylpyrrolidone (PVP) (Sigma, St. Louis, MO, USA) blocking buffer (6.25 mM PVP, 18.8 mM NaCl in 50 mL PBS-Tween) overnight at 4 °C with shaking. On the following day, the membrane was incubated with anti-C9 primary mAb diluted in blocking buffer (10 ng/mL), overnight at 4 °C with shaking. Next, the membrane was washed 5 times with PBS-Tween. After washing, secondary antibodies were added, species-specific Goat anti-Mouse horseradish peroxidase (GAM-HRP (Invitrogen, Carlsbad, CA, USA)) diluted to 1:2500 in PBS buffer, then the membrane was incubated overnight at 4 °C with shaking. Next, the membrane was washed 5 times with PBS-Tween. The membrane was developed with Pierce ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s guide and visualized with GelLogic 2200Pro (Carestream, Rochester, NY, USA).
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