Cells and skeletal muscle samples were lysed in RIPA Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2mM EDTA, 1 % Triton X100, 0.5 % sodium deoxycholate) supplemented with protease (Complete, Roche) and phosphatase (PhosSTOP, Roche) inhibitor cocktails. Lysates were cleared by centrifugation at 14,000 × g for 20 min at 4 °C. Protein concentrations were determined using BCA Assay. Equal amounts of protein (20 μg) were separated by SDS-PAGE, transferred onto Protran Nitrocellulose Membranes (GE Healthcare) using eBlot L1 Fast Wet Transfer System (GenScript), followed by incubation with indicated primary antibodies against α-actinin (R&D, MAB9830; 1:1000), LC3 (Abcam, ab192890; 1:1000), MuRF1 (Santa Cruz, sc-398608; 1:1000), MuRF2 (Abcam, ab4387; 1:1000), and γ-tubulin (Sigma-Aldrich T-5326; 1:1000). Signals of anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:10’000) were visualized on a LAS-3000 Luminescent Image Analyzer (Fujifilm) using Lumi-Light Western Blotting Substrate (Roche).
Eblot l1 fast wet transfer system
Manufactured by GenScript
Sourced in United States
The EBlot L1 Fast Wet Transfer System is a laboratory equipment designed for the fast and efficient transfer of proteins from polyacrylamide gels to membranes. It features a compact and user-friendly design to facilitate the western blotting process.
Automatically generated - may contain errors
Lab products found in correlation
Las 3000 luminescent image analyzer, by Fujifilm (2 mentions)
Protran nitrocellulose membrane, by GE Healthcare (2 mentions)
Phosstop, by Roche (2 mentions)
Lumi light western blotting substrate, by Roche (2 mentions)
Protran, by Cytiva (2 mentions)
Ab192890, by Santa Cruz Biotechnology (1 mentions)
Sc 398608, by Abcam (1 mentions)
T5326, by Merck Group (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Beyoecl star kit, by Beyotime (1 mentions)
Ab170904, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Imagequant las4000 luminometer, by GE Healthcare (1 mentions)
Chemiluminescence imaging system, by Clinx (1 mentions)
6 protocols using eblot l1 fast wet transfer system
1
Immunoblotting Analysis of Skeletal Muscle Proteins
2
Quantitative Western Blot Analysis
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Tissues or cells were lysed by RIPA buffer (Thermo Fisher) supplemented with protease and phosphatase inhibitors (Thermo Fisher). Lysates were sonicated and then cleared by centrifugation at 12,000 g for 15 min. Protein concentrations were quantified with a BCA Protein Assay kit (Beyotime Biotechnology). Equal amounts of proteins were separated by SurePAGETM precast polyacrylamide gels with a gradient between 4 and 20% (GenScript) and blotted onto 0.22 μm PVDF membranes (Millipore) by eBlot L1 Fast Wet Transfer System (GenScript). After blocking in TBST containing 5% skim milk for 1 h at room temperature, membranes were incubated with appropriate primary antibodies overnight at 4°C on a shaker. Specific banding was detected by incubation with appropriate HRP-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) system (Thermo). Band intensities were quantified using ImageJ software. Detailed antibody information is provided in Supplementary Table 1 .
3
Phospho-STAT3 Western Blot Analysis
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Cells were lysed in RIPA Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X100, 0.5% sodium deoxycholate) supplemented with protease (Complete, Roche) and phosphatase (PhosSTOP, Roche) inhibitor cocktails. Lysates were cleared by centrifugation at 14 000g for 20 minutes at 4 °C. Protein concentrations were determined using BCA Assay. Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto Protran nitrocellulose membranes (GE Healthcare) using eBlot L1 Fast Wet Transfer System (GenScript), followed by incubation with the indicated primary antibodies against Phospho-Stat3 (Cell Signaling Technology Cat# 9131, RRID:AB_331586; 1:1000), and γ-tubulin (Sigma-Aldrich Cat# T5326, RRID:AB_532292; 1:1000). Signals of antirabbit (Millipore Cat# 401393-2ML, RRID:AB_437797) and antimouse (Millipore Cat# 401253, RRID:AB_437779) IgG horseradish peroxidase–conjugated secondary antibody (1:10 000) were visualized on a LAS-3000 Luminescent Image Analyzer (Fujifilm) using Lumi-Light Western Blotting Substrate (Roche).
4
Western Blot Analysis of Brain Capillary Proteins
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Isolated brain capillaries or hCMEC/D3 cells were homogenized in cold RIPA lysis buffer (Beyotime Biotechnology) containing phenylmethylsulfonyl fluoride. Samples were mixed with 5× lithium dodecyl sulfate sample buffer and 10% DTT reducing agent. The samples were run in 4–20% Tris-Gly gradient gels and transferred to the eBlot® L1 Fast Wet Transfer System (Genscript, USA). Membranes were blocked for 1 h and incubated overnight at 4°C with primary antibodies against GAPDH (1:5000, ab8226, Abcam), TET2 (1:500, #18,950, Cell Signaling), or P-gp (1:1000, ab170904, Abcam). The membranes were washed and incubated with horseradish peroxidase–conjugated secondary IgG (1:5000; SGARHAP, Yishan Biotech) for 1 h at RT. Protein bands were visualized using a BeyoECL Star Kit (Beyotime) and an ImageQuant LAS 4000 luminometer (GE, USA). The optical density of the protein bands was measured with ImageJ software (NIH, Bethesda, MD, USA).
5
Detecting Influenza Viral Proteins by Western Blot
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For western blot, 1 μg of H1N1, H3N2, H5N1, and H7N9 NA recombinant proteins in reducing SDS loading buffer were heated in boiling water for 10 min and subjected to 4%–12% SDS PAGE. Separated proteins were transferred to a polyvinylidene fluoride (PVDF) blotting membrane (Merck, Germany, IPVH00010) using the eBlot L1 Fast Wet Transfer System (GenScript, USA). The blotted membrane was blocked with 5% skim milk in TBST (0.1% Tween 20 in Tris-buffered saline [TBS]) for 1 h and incubated with 5 μg/mL of FNA1 mAb in TBST containing 5% skimmed milk at 4°C overnight. After washing with TBST, the membrane was incubated with anti-human IgG secondary antibody for 1 h. After washing the membrane three times, the entire membrane was covered with an enhanced chemiluminescence substrate (PerkinElmer, USA, NEL105001EA), and images were acquired using a Chemiluminescence Imaging System (Clinx Science Instruments Co., China).
6
Rapid Protein Separation and Transfer
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4–12% SurePAGE, Bis–Tris Gel, 12 well, 80 µl (GenScript – M00653) were used for protein separation. MES Buffer Powder packs (GenScript – M00677) were reconstituted in one liter of ddH2O for gel separation in GenBox Mini Tank (GenScript – L00780).
The Complete eBlot L1 Transfer sandwich (PVDF) (GenScript – L00727) was used in conjunction with the eBlot L1 Fast Wet Transfer System (GenScript – L00686) using the preset “Standard” setting for all transfers requiring 16 min per membrane.
PVDF membranes were activated in 100% Ethanol with gentle shaking for approximately five to ten minutes and then equilibrated in eBlot L1 PVDF Membrane Equilibration Buffer (1X) for about 2–5 min prior to transfer. After protein separation, the gels were incubated in water for about two minutes prior to transfer.
The Complete eBlot L1 Transfer sandwich (PVDF) (GenScript – L00727) was used in conjunction with the eBlot L1 Fast Wet Transfer System (GenScript – L00686) using the preset “Standard” setting for all transfers requiring 16 min per membrane.
PVDF membranes were activated in 100% Ethanol with gentle shaking for approximately five to ten minutes and then equilibrated in eBlot L1 PVDF Membrane Equilibration Buffer (1X) for about 2–5 min prior to transfer. After protein separation, the gels were incubated in water for about two minutes prior to transfer.
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