Proteins were extracted from HEK 293 T cells or human prefrontal cortex brain tissues using tissue total protein lysis buffer (Beyotime, Shanghai, China) for each assay. Proteins were electrophoretically separated on SDS-PAGE gels and subsequently immunoblotted onto nitrocellulose membranes that were blocked in skim milk and incubated at 4 °C for 16 h using antibodies against GFP (1:5000, ab290, Abcam), β-tubulin (1:10,000, BS1482M, Bioworld), TARP γ-8 (1:500, abs132666a, Absin) and GAPDH (1:10,000, 60,004–1-Ig, Proteintech). Detection was carried out using the horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG (1:10,000, BS12478, Bioworld) and goat anti-rabbit IgG (1:15,000, BS13278, Bioworld) along with a High-sig ECL kit (Tanon, Shanghai, China), and signals were recorded using a Tanon 5200 multi automatic chemiluminescence image analysis system (Tanon, Shanghai, China). For western blot quantitation, images were analyzed using ImageJ and expressed as arbitrary units (AU). GFP protein expression was normalized to β-tubulin protein expression, and TARP γ-8 expression was normalized to GAPDH expression.
Tissue total protein lysis buffer
Manufactured by Beyotime
Tissue total protein lysis buffer is a solution used to extract and solubilize total proteins from tissue samples. It is designed to efficiently disrupt cells and denature proteins, enabling the subsequent quantification and analysis of the total protein content.
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2 protocols using tissue total protein lysis buffer
1
Protein Extraction and Western Blot Analysis
2
Rat Model of Neuronal Apoptosis
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A total of 80 male specific-pathogen-free Sprague-Dawley rats (age, 8–9 weeks; weight, 220–260 g; mean weight, 240 g) were obtained from the Department of Animal Center, Medical College of Nantong University. All animals were raised in a temperature- and humidity-controlled room (22±1°C; 55–65%) with a 12-h light/dark cycle and food and water were available ad libitum. Primary antibodies against pro caspase-3 (cat. no. ab32499), cleaved caspase-3 (cat. no. ab32042), NeuN (cat. no. ab279290), ret proto-oncogene (RET; cat. no. ab134100) and β-tubulin (cat. no. ab18207), secondary goat anti-rabbit IgG H&L (cat. no. ab6721) and rabbit anti-mouse IgG (cat. no. ab6728) and Hoechst 33342 Staining Dye Solution (cat. no. ab228551) were purchased from Abcam. The ECL Western Blot Substrate (cat. no. ab65623) were purchased from Abcam. The secondary Alexa Fluor®−488 (anti-mouse; cat. no. A-11029) and Alexa Fluor®−568 (anti-rabbit; cat. no. A-11036) antibodies were purchased from Thermo Fisher Scientific, Inc. The BCA protein quantitation kit, tissue total protein lysis buffer, PMSF and fluorescent sealing solution were from Beyotime Institute of Biotechnology; PVDF membrane and protease inhibitor were from Roche Diagnostics. Blotting paper was purchased from Bio-Rad Laboratories, Inc.
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