Cells were washed in PBS 1× and frozen dry at -80 °C or lysed directly in lysis buffer (10 mM Tris–HCl pH7.6, 150 mM NaCl, 1% Triton X100, 1 mM EDTA, 0.1% deoxycholate, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromophenol blue) and boiled. The lysates were resolved by SDS-PAGE and analyzed by immunoblotting. Incubation with a primary FLAG antibody coupled to horseradish peroxidase (HRP) (mouse monoclonal M2, Sigma-Aldrich) and primary Myc coupled to HRP (Sigma-Aldrich) or Actin (Sigma-Aldrich) antibody followed by an HRP-conjugated secondary antibody was performed. Bioluminescence was then measured (Clarity ECL Western Blotting Substrate, Bio-Rad) using a ChemiDoc system (Bio-Rad).
Mouse monoclonal m2
Manufactured by Merck Group
Mouse monoclonal M2 is a laboratory reagent used in various research applications. It is a mouse-derived antibody that can be used to detect and identify specific target proteins or molecules in a sample. The core function of this product is to serve as a tool for researchers to analyze and study their samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc system, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Amersham hybond p, by GE Healthcare (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Roti quant, by Carl Roth (1 mentions)
Hybond, by Cytiva (1 mentions)
2 protocols using mouse monoclonal m2
1
Western Blot Analysis of Protein Expression
2
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial cells or partly purified protein fractions were supplied with 6× Laemmli sample buffer to a final concentration of 1× sample buffer (63 (link)). COS-7 cells were transfected for 48 h (12-well plates) with TBX expression constructs, trypsinized, and resuspended in PBS. After pelleting (5 min, 13,000 rpm in an Eppendorf microcentrifuge) cells were resuspended in 25–50 μl urea lysis buffer (7 M urea, 2 M thiourea, 5 mM DTT, 2% Chaps, 0.01% Proteinase Inhibitor Cocktail (Sigma-Aldrich P8340), 1 mM PMSF). Cells were lysed for 30 min on ice. Protein concentration was determined with a Bradford assay (Roti-Quant, Roth, Karlsruhe, Germany). Appropriate volumes of cell lysate were combined with 6× Laemmli sample buffer and loaded on an 8 or 10% SDS gel. PageRuler™ Prestained Protein Ladder (Thermo Scientific) was used to calibrate gel mobilities. Blotting to a PVDF membrane (Amersham Hybond™-P, GE Healthcare, München, Germany) and detection procedure was as described above in the EMSA protocol. FLAG epitope was detected with mouse monoclonal M2 (1:1000–2000), alpha-tubulin with mouse monclonal B5-1-2 (1:1000) (both Sigma-Aldrich). HRP-tagged goat anti-mouse IgG was used as a secondary antibody (1:10,000, Jackson ImmunoResearch, Dianova, Hamburg, Germany).
For pixel density analysis of fluorographs, several exposures were taken and analyzed using the ImageJ program2 .
For pixel density analysis of fluorographs, several exposures were taken and analyzed using the ImageJ program
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!