Cells were washed with one volume of PBS and collected by scraping in PBS. Cell suspensions were centrifuged at 5000×g for 5 min at 4 °C. Pellets were lysed in buffer containing Tris 10 mM, pH 7.4, NaCl 150 mM, EDTA 1 mM, EGTA 1 mM, Triton X-100 1% v/v, NP-40 0.5% v/v, and Halt Protease Inhibitor Cocktail (Thermo). Protein concentrations were determined by BCA Protein Assay (Thermo). Lysates were normalized for protein concentration and subjected to SDS-PAGE separation on stain-free tris-glycine gels (Bio-Rad), cross-linked and imaged with the Chemidoc system (Bio-Rad), transferred to PVDF membranes with the Trans-Blot Turbo transfer system (Bio-Rad), imaged, blocked in 5% blocking buffer (Bio-Rad), blotted in primary and secondary antibodies, and developed using WesternBright ECL (Advansta). Band signal intensity was normalized to total protein per lane as determined from the stain-free gel or membrane images.
Stain free tris glycine gels
Manufactured by Bio-Rad
Stain-free tris-glycine gels are a type of electrophoresis gel used for the separation and analysis of proteins. These gels are designed to be used without the need for traditional protein staining methods, allowing for a more efficient and streamlined analytical process.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc system, by Bio-Rad (1 mentions)
Westernbright ecl, by Advansta (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Image lab software package, by Bio-Rad (1 mentions)
3 protocols using stain free tris glycine gels
1
Protein Extraction and Western Blot Analysis
2
Protein Profiling via Size Exclusion Chromatography
3
Quantification of Protein Levels by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cell pellets were collected following the indicated treatments and time points and normalized by units OD600 through dilution in Laemmli buffer. Diluted samples were boiled at 98°C for 10 min and loaded into Stain‐Free Tris‐glycine gels (Bio‐Rad) for separation by SDS‐PAGE and transfer to a nitrocellulose membrane as per manufacturer guidelines. Membranes were blocked for 1 h in 5% skim milk powder in TBST and protein levels were detected using either anti‐DnaK antibody (Schramm et al., 2017 ) or the anti‐GFP antibody (Thermo Fisher Scientific, #A‐11122). A secondary anti‐rabbit HRP‐conjugated antibody was used to detect primary antibody binding, and SuperSignal Femto West (Thermo Fisher Scientific) used to develop the membrane. Blots were scanned using a Chemidoc (Bio‐Rad) system and signal quantification was performed using the Image Lab software package (Bio‐Rad).
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