Pancreatic ER fractions as well as total tissue lysates were examined by Western blot as follows: ER fractions (10–20 μg) were separated on Invitrogen Tris-glycine sodium dodecyl sulfate–polyacrylamide gel electrophoresis 4%–20% gradient gels, transferred to nitrocellulose membranes using Bio-Rad (Hercules, CA) TransBlot Turbo rapid transfer system (15 minutes at a constant current of 1.25 A per minigel), and blocked with 5% bovine serum albumin in Tris-buffered saline buffer with 0.075% Tween 20. Subsequent incubations and washes were performed in Tris-buffered saline with 0.075% Tween 20. Primary antibodies used against lipase, carboxyl ester lipase (Cel) (sc-34878), signal recognition particle 72 were from Santa Cruz Biotechnologies (Santa Cruz, CA). Extracellular signal-regulated kinase 1/2 antibody was from Cellular Signaling Technologies (Beverly, MA). ActivX Desthiobiotin-FP Serine Hydrolase Probe and streptavidin–horseradish peroxidase were from Thermo Fisher Scientific. ER fractions (50 μg) were incubated with probe (2 μg/mL) in 10 mmol/L Tris-HCl, pH 7.4 for 20 minutes at room temperature, then Triton X-100 (Bio-Rad) was added to 1% and the clarified mixture was incubated for a further 20 minutes at room temperature. Samples then were prepared by addition of gel loading buffer and further processed for Western blot as described earlier.
Trans blot turbo rapid transfer system
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo rapid transfer system is a laboratory equipment designed for fast and efficient protein transfer from polyacrylamide gels to membranes. The system utilizes a proprietary technology to enable rapid transfer of proteins, ensuring reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (4 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Triton x 100, by Bio-Rad (1 mentions)
Streptavidin horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Activx desthiobiotin fp serine hydrolase probe, by Thermo Fisher Scientific (1 mentions)
Lowry protein assay, by Bio-Rad (1 mentions)
Criterion gel system, by Bio-Rad (1 mentions)
Infrared dye conjugated secondary antibody, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Odysseyxl system, by LI COR (1 mentions)
Epac1, by Abcam (1 mentions)
Epac2, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Proteintech (1 mentions)
8 protocols using trans blot turbo rapid transfer system
1
Western Blot Analysis of Pancreatic ER Fractions
2
Quantitative Western Blot Protein Analysis
3
Protein Quantification and Characterization
4
Western Blot Protein Analysis Protocol
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The supernatants were removed, and the cells were washed with PBS three times. Then, the cells were lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) supplemented with 5× SDS-PAGE sample loading buffer and heated at 95 °C for 10 min. Then, the proteins were separated on SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes by a Trans-Blot Turbo rapid transfer system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The membranes were blocked in Tris-buffered saline (TBS) supplemented with 5% milk for 2 h at room temperature and then incubated with a primary antibody at 4 °C overnight. The membranes were then washed in TBS supplemented with 0.1% Tween 20 and incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. The membranes were washed and imaged.
5
Western Blot Analysis Protocol
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For western blot analysis, cells were lysed in RIPA buffer (Beyotime, Shanghai, China) and denatured by adding 4× Laemmle SDS-PAGE buffer (containing DL-dithiothreitol), followed by heating for 15 min at 100 °C. The proteins were then separated on SDS-PAGE gels and transferred onto nitrocellulose membranes using a Trans-Blot Turbo rapid transfer system (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. The membranes were blocked in 5% defatted milk (dissolved in Tris-buffered saline (TBS)) for 1 h at 37 °C and then incubated with a primary antibody for 1 h at room temperature or overnight at 4 °C. The membranes were then washed three times (5 min per time) using a wash buffer (TBS containing 0.1% Tween 20) and incubated with an IRDye® 800CW secondary antibody for 1 h at 37 °C. The membranes were washed three times in wash buffer and imaged using an Odyssey Imaging System (LI-COR, USA) to visualize the protein bands. α-Tubulin was used as the loading control.
6
Western Blot Protein Analysis Protocol
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For the Western blot analysis, cells were lysed in RIPA buffer (Beyotime) and denatured by adding 4× Laemmli SDS-PAGE buffer (containing DL-Dithiothreitol), followed by heating for 10 min at 100 °C. The proteins were then separated on SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes by a Trans-Blot Turbo rapid transfer system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The membranes were blocked in 5% defatted milk (dissolved in Tris-buffered saline (TBS)) for 1 h at room temperature and then incubated with a primary antibody for 1 h at room temperature or overnight at 4 °C. The membranes were then washed extensively in wash buffer (TBS containing 0.1% Tween 20) three times (for 5 min each time) with agitation and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech) for 1 h at room temperature according to the species source of the primary antibody. The membranes were washed three times in wash buffer and imaged using an enhanced chemiluminescence substrate solution (Sigma-Aldrich, P90720) to visualize the protein bands. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or α-Tubulin was utilized as a loading control.
7
Western Blot Protein Analysis Protocol
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For western blot analysis, cells were lysed in RIPA buffer (Beyotime) and denatured by adding 4× Laemmli sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer (containing DL-dithiothreitol), followed by heating for 15 min at 100°C. The proteins were then separated on SDS-PAGE gels and transferred onto nitrocellulose membranes using a Trans-Blot Turbo rapid transfer system (Bio-Rad), according to the manufacturer’s instructions. The membranes were blocked in 5% defatted milk (dissolved in Tris-buffered saline (TBS)) for 1 h at 37°C and then incubated with a primary antibody for 1 h at 25°C or 12 h at 4°C. The membranes were then washed thrice (5 min per wash) using a wash buffer (TBS containing 0.1% Tween-20) and incubated with an IRDye® 800CW secondary antibody for 1 h at 37°C. The membranes were washed thrice in wash buffer and imaged using an Odyssey Imaging System (LI-COR, USA) to visualize the protein bands. α-Tubulin was used as a loading control.
8
Western Blot Analysis of Cellular Proteins
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For the Western blot analysis, cells were lysed in RIPA buffer (Beyotime, Shanghai, China) and denatured by adding 4× Laemmle SDS-PAGE buffer (containing DL-Dithiothreitol), followed by heating for 15 min at 100 °C. The proteins were then separated on SDS-PAGE gels and transferred onto NC membranes by a Trans-Blot Turbo rapid transfer system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The membranes were blocked in 5% defatted milk (dissolved in Tris-buffered saline (TBS)) for 1 h at 37 °C and then incubated with a primary antibody for 1 h at room temperature or overnight at 4 °C. The membranes were then washed extensively in wash buffer (TBS containing 0.1% Tween 20) three times (for 5 min each time) with agitation and incubated with an IRDye 800CW secondary antibody for 1 h at 37 °C according to the species source of the primary antibody. The membranes were washed three times in wash buffer and imaged using an Odyssey (LICOR, Lincoln, NE, USA) to visualize the protein bands. α-Tubulin was utilized as a loading control.
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