All western blots were performed on cells prior to their use for in vivo experiments to verify the level of protein depletion following the different treatments. Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (25 mM Tris HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS), sonicated, and quantified using DC™ Protein Assay (Biorad). Sixty micrograms of total protein were incubated at 95°C for 5 min in Laemmli 4X (Bio-Rad), and loaded onto a 6% or 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Bio-Rad) using wet transfer at 120V for 1.5 hours. Ponceau S solution (sigma P7170–1L) was performed for each blot to validate even transfer across the membrane. Membranes were blocked with 1% bovine serum albumin (Fisher BP1600–100) in 1X TBS-T and probed with primary antibody overnight. Membranes were then washed in 1X TBS-T and incubated with the corresponding LICOR secondary antibody (1:15,000 dilution) for 1 hour at room temperature, and signals were acquired and quantified with the Odyssey system (LI-COR Biosciences).
Laemmli 4x
Manufactured by Bio-Rad
Sourced in United States
Laemmli 4X is a concentrated sample buffer used in the preparation of protein samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It contains the necessary components to denature and solubilize proteins, allowing for their separation and analysis based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey system, by LI COR (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Bp1600 100, by Thermo Fisher Scientific (2 mentions)
Clone 36 e cadherin, by BD (1 mentions)
Sc 516102, by Santa Cruz Biotechnology (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
3 protocols using laemmli 4x
1
Western Blot Protein Quantification Protocol
2
LNA-AMOs Modulate E-cadherin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed to analyze the effect LNA-AMOs on the expression E-cadherin protein, under the same conditions stated above for the qRT-PCR analysis. MKN74 cells, treated or not with LNA-AMOs were washed three times with ice-cold PBS, and then disrupted using a lysing buffer, containing 1% Triton X-100 (v/v), 1% NP-40 (v/v, pH 7.4), supplemented with phosphatase inhibitor (1: 100) and protease inhibitor (1: 7) during 15 min. Samples were further centrifuged at 14,000 rpm for 20 min at 4 °C and the supernatants collected. 20 μg of total protein were mixed with laemmli 4x (Bio-rad, CA, USA), denatured at 95 °C for 5 min, and separated on a 10% polyacrylamide gel. Proteins were then transferred onto a nitrocellulose membrane and the membranes were blocked 30 min at RT with 5% dry milk in 0.5% Tween-PBS, followed by incubation at RT during 2 h with the antibodies in the following dilutions: a mouse monoclonal anti-α-E-Cadherin antibody (1: 1000) (Clone 36/E-Cadherin, BD Biosciences, CA, USA), and a mouse anti-α-GAPDH antibody (1: 30,000) (sc-47724, Santa Cruz Biotechnology, TX, USA). Blots were then washed 3 times with 0.5% PBS-Tween and incubated with a secondary anti-mouse antibody (1: 20,000) (sc-516102, Santa Cruz Biotechnology) for 2 h at RT.
3
Western Blot Protein Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All western blots were performed on cells prior to their use for in vivo experiments to verify the level of protein depletion following the different treatments. Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (25 mM Tris HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS), sonicated, and quantified using DC™ Protein Assay (Biorad). Sixty micrograms of total protein were incubated at 95°C for 5 min in Laemmli 4X (Bio-Rad), and loaded onto a 6% or 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Bio-Rad) using wet transfer at 120V for 1.5 hours. Ponceau S solution (sigma P7170–1L) was performed for each blot to validate even transfer across the membrane. Membranes were blocked with 1% bovine serum albumin (Fisher BP1600–100) in 1X TBS-T and probed with primary antibody overnight. Membranes were then washed in 1X TBS-T and incubated with the corresponding LICOR secondary antibody (1:15,000 dilution) for 1 hour at room temperature, and signals were acquired and quantified with the Odyssey system (LI-COR Biosciences).
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!