Samples were separated on native 4–12% Tris-Glycine gels in Tris-Glycine running buffer at pH 8.3 (Invitrogen, Darmstadt, Germany). The gels were incubated in 0.1% SDS for 15 min and the proteins were transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) for 80 min at 200 mA. The transferred proteins were fixed to the membranes by incubation in 10% acetic acid for 15 min and air drying. After rehydration in 1× Tris-buffered saline (TBS) the membranes were treated with blocking solution (10% non-fat dry milk in 1× TBS) for 1 h at RT, incubated either with anti-RPA70 (sc-166023, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-Tipin (sc-160865, Santa Cruz Biotechnology) primary antibody for 1 h at RT and washed twice with TBS containing 0.05% Tween 20 (TBS-T) and once with TBS. Membranes were further incubated with horseradish peroxidase-conjugated anti-mouse or anti-goat secondary antibodies (Santa Cruz Biotechnology) for 1 h at RT and washed twice with TBS-T and once with TBS. Bound antibodies were detected by chemiluminescence using ECL reagents (GE Healthcare, Freiburg, Germany) and an ImageQuant LAS 4000 imager (GE Healthcare).
Tris glycine running buffer
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Tris-Glycine running buffer is a common buffer solution used in gel electrophoresis to separate proteins or nucleic acids based on their size or charge. It provides a consistent pH and ionic environment to facilitate the migration of samples through the gel matrix during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 3 8 tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin standard, by Thermo Fisher Scientific (1 mentions)
Bicinchonic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phosstop phosphatase inhibitor, by Merck Group (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
4 protocols using tris glycine running buffer
1
Western Blot Analysis of RPA70 and Tipin
2
ACE2 Expression Quantification under Shear Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantification of ACE2 expression was performed using lysates extracted from cell monolayers cultured on hydrogels exposed to 24 h of fluid shear stress applied by a cone and plate rheometer. 50 mM SARS-CoV-1 subunit S1 was added during the final 3 h of flow. Cell lysates were separated using a tris–glycine gel (Invitrogen) in tris–glycine running buffer (Invitrogen). Separated protein was transferred to 0.45-μm PVDF membrane (Invitrogen). The membrane was probed over night with ACE2 (Santa Cruz Biotech) (1:250) and beta-Actin (1:400) (Santa Cruz Biotech) antibodies and visualized with anti-mouse HRP conjugated secondary antibodies (1:4000) (Azure).
3
Native PAGE analysis of BMDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were stimulated as described and lysed in RIPA lysis buffer without SDS. Lysates were centrifuged at 2,300 ×g for 10 min to pellet DNA. Supernatant was centrifuged at 16,100 ×g for 25 min, and the pellet was resuspended in native PAGE sample buffer (Thermo Fisher Scientific). The samples were loaded onto NuPage 3%–8% Tris-acetate gels (Thermo Fisher Scientific) without boiling, and native PAGE was conducted using Tris-glycine running buffer (Thermo Fisher Scientific). The gel was soaked in 10% SDS solution for 10 min before performing semidry transfer and continuing with conventional immunoblot.
4
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and lysed with lysis buffer (150 mM Hepes (pH 7.5), 150 mM NaCl, 100 mM NaF, 10 mM ethylene diamine tetraacetic acid (EDTA), 10 mM Na4P2O7, 1% Triton-X-100, 0.1% SDS, EDTA-free protease inhibitor (Thermo Fisher Scientific; # 88266), PhosSTOP phosphatase inhibitor (Sigma-Aldrich; # 04906837001)). Lysates were incubated on ice for 30 min and centrifuged for 5 min at 8000× g. Protein concentration of the supernatant was determined using a bicinchonic acid protein assay kit and bovine serum albumin standards (Thermo Fisher Scientific; # 23225). Proteins were then separated on 10–20% Tris-glycine gels (Thermo Fisher Scientific) using Tris-glycine running buffer (Thermo Fisher Scientific; # LC2675). Proteins were transferred to polyvinylidene difluoride membrane in running buffer (25 mM Trizma base, 192 mM glycine) containing 10% methanol and blocked in 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween. Membranes were incubated with primary antibodies overnight at 4 °C, and horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were detected using Clarity Western Enhanced Chemiluminescence (ECL) Substrate (Biorad, Temse, Belgium; # 170-5061) or ECL Western Blotting Substrate (Thermo Fisher Scientific; 32106). Individual experiments were always performed in duplicate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!