Western blot analysis was performed as described previously 14 (link). Cells were lysed in PRO-PREP protein extract solution (iNtRON Biotechnology, Houston, TX, USA) to obtain total cell lysates, and the lysates were centrifuged at 100,000 × g for 20 min at 4°C. Protein concentrations were determined using the Bradford method. For preparation of sample loading, equal volumes of 2× sodium dodecyl sulfate sample buffer (0.1 mol/L Tris-HCI, 20% glycerol, 4% sodium dodecyl sulfate, and 0.01% bromophenol blue) and supernatant fractions from the lysates were mixed. Proteins (60 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 90 min at 110 V. The separated proteins were transferred onto polyvinylidene difluoride membranes for 2 h at 20 mA using SD Semi-dry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the membranes using 5% nonfat milk in Tris-buffed saline (pH 7.0), the membranes were incubated overnight at 4°C with primary antibodies (anti-phospho-MYPT1 and anti-MYPT1 antibodies) at a dilution of 1:500 in 5% skim milk in Tris-buffed saline containing Tween-20. Bound antibody was detected with horseradish peroxidase-conjugated anti-mouse IgM. Membranes were washed and developed using the Western Blotting Luminol Reagent system (iNtRON Biotechnology) and autoradiography.
Sd semi dry transfer cell
Manufactured by Bio-Rad
Sourced in United States
The SD Semi-dry Transfer Cells are designed for the efficient transfer of proteins from polyacrylamide gels to membranes. The cells feature a simple, easy-to-use design that ensures consistent and reliable protein transfer.
Automatically generated - may contain errors
Lab products found in correlation
Pro prep protein extract solution, by iNtRON Biotechnology (2 mentions)
Luminol reagent system, by iNtRON Biotechnology (2 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Bcip nbt substrate solution, by Merck Group (1 mentions)
Anti phospho p44 42 erk, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Immobilon p polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
6 protocols using sd semi dry transfer cell
1
Western Blot Analysis of MYPT1 Phosphorylation
2
Western Blot Analysis of Protein Extracts
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Western blot analysis was performed using the method described by Lee et al. 20 (link). Cells were lysed in PRO-PREP protein extract solution to obtain total cell lysates, and the lysates were centrifuged at 13,000 rpm for 20 min at 4°C. The protein concentrations were determined using the Bradford method. For sample loading, equal volumes of 2× sodium dodecyl sulfate sample buffer (0.1 mol/L Tris-HCI, 20% glycerol, 4% sodium dodecyl sulfate and 0.01% bromophenol blue) and supernatant fractions from the lysates were mixed. Proteins (60 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 90 min at 110 V. The separated proteins were transferred to polyvinylidene difluoride membranes for 2 h at 20 mA using SD Semi-dry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the membranes with 5% nonfat milk in Tris-buffered saline (pH 7.0), the membranes were incubated overnight at 4°C with specific antibodies at a dilution of 1:500 in 5% skim milk in Tris-buffered saline containing Tween-20. Bound antibody was detected with horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG. The membranes were washed and developed using a western blotting luminol reagent system (iNtRON Biotechnology, Houston, TX, USA) and autoradiography.
3
Western Blot Analysis Protocol
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Western blot analysis was performed as described by Ok et al. 24 (link). In brief, the cells were lysed in PRO-PREP protein extract solution (iNtRON Biotechnology, Houston, TX, USA) for total cell lysates, and the lysates were centrifuged at 100,000 × g for 20 min at 4°C. The protein concentration was determined using the Bradford method. For the preparation of the sample loading, equal volumes of 2× sodium dodecyl sulfate (SDS) sample buffer (0.1 mol/L Tris-HCI, 20% glycerol, 4% SDS, and 0.01% bromophenol blue) and supernatant fractions from the lysates were mixed. Sixty micrograms of protein were separated with 10% SDS-polyacrylamide gel electrophoresis for 90 min at 110 V. The separated proteins were transferred to polyvinylidene difluoride membranes for 2 h at 20 mA using SD Semi-dry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the membranes in 5% nonfat milk in Tris-buffed saline (pH 7.0), the membranes were incubated overnight at 4°C with specific antibodies at a dilution of 1:500 in 5% skim milk in Tris-buffed saline containing Tween-20. Bound antibody was detected with horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG. Membranes were washed and developed using a western blotting luminol reagent system (iNtRON Biotechnology) and autoradiography.
4
Cadmium-Induced Signaling Pathway Analysis
5
Immunodetection of Cry1Ab Toxin in Transgenic Tomatoes
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Western immunoassay was performed with cell-free extracts of transgenic tomato plants expressing Cry1Ab toxin as described earlier (Sambrook et al. 1989 ). Aliquots of the total plant protein samples were boiled for 10 min with 2× sample loading dye [50 mM Tris–HCl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol] and electrophoresed on 10% denaturing SDS-PAGE. The resolved protein bands on SDS-PAGE were transferred onto immunoblot™ PVDF membrane (Bio-Rad, USA) using SD semi-dry transfer cell (Bio-Rad, USA) in transfer buffer [25 mM Tris base, 192 mM glycine (pH 8.3) and 0.1% SDS]. The membranes were blocked for 2 h at 25°C in blocking buffer and incubated with primary antibody (rabbit polyclonal to B. thuringiensis Cry1Ab toxin protein, Agdia, USA) diluted to 1:1000 ratio in blocking buffer, for 2 h at 25°C. The membranes were washed four times with PBST, followed by incubation with secondary antibody (goat polyclonal to rabbit IgG-alkaline phosphatase conjugated antibody, Sigma, USA) at 1:5000 dilution for 2 h at 25°C and colour development with BCIP-NBT substrate solution (Sigma, USA).
6
Western Blot Analysis of Signaling Pathways
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Total cell lysates were extracted using CytoBuster™ Protein Extraction Reagent (Novagen). Equal amounts of protein were separated on 10 to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels at 300 mA for 20 min. They were then transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Billerica, MA, USA) using a trans-blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). After transfer, the membranes were incubated in Tris buffered saline (TBS), 5% dry milk, and 0.2% Tween 20 for 1 h. They were then further incubated in TBS and 0.2% Tween 20 with specific antibodies at 4 °C overnight. After washing, a horseradish peroxidase (HRP)-labeled secondary antibody was applied and the membranes were incubated for 2 h. Immunoblot detection was performed using Immobilon Western HRP Substrate (Millipore Sigma). The following antibodies from Cell Signaling Technology were used: anti-protein kinase B (PBK/Akt), anti-phospho-Akt (Ser473), anti-c-jun N-terminal kinase (JNK), anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-p44/42 extracellular signal-related kinase (ERK), anti-phospho-p44/42 ERK, and anti-GAPDH.
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