This protocol was standarized in our lab based on the publication of Ahn T et al (2001):\u00a0 Polyacrylamide Gel Electrophoresis without a stacking Gel: Use of Amino Acids as Electrolytes. DOI:10.1006\/abio.2001.5038\nThese gels run similar to a gradient gel: In a 6% gel you will be able to separate proteins ranging from 270 kDa to 25 kD an dhave them in the same gel.The adaptation to Stain-Free system was done based on the publication by Ladner CL et al (2004) : Visible fluorescent detection of proteins in polyacrylamide gels without staining. DOI:10.1016\/j.ab.2003.10.047We have applied this protocol in the following publications: Zeitler AF, Gerrer KH, Haas R, Jim\u00e9nez-Soto LF. Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology. J Microbiol Methods. 2016 Jul;126:38-41. doi: 10.1016\/j.mimet.2016.04.016. PubMed PMID: 27150675.Jim\u00e9nez-Soto LF, Haas R. The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated. Sci Rep. 2016 Mar 17;6:23227. doi: 10.1038\/srep23227. PubMed PMID: 26983895; PubMed Central PMCID: PMC4794710.Jim\u00e9nez-Soto LF, Clausen S, Sprenger A, Ertl C, Haas R. Dynamics of the Cag-type IV secretion system of Helicobacter pylori as studied by bacterial co-infections. Cell Microbiol. 2013 Nov;15(11):1924-37. doi: 10.1111\/cmi.12166. PubMed PMID: 23844976.