b'Troubleshooting tipsSample preparationProtein gel electrophoresisObservationPossible causeSuggested solution Protein bands loseReduce the sample loads. The maximum resolution, lanesToo much protein loaded per lane recommended sample load for optimal resolution in have streaks and aremini gels with 10, 12, 15, or 17 wells is 0.5 g per band not straight or about 1015 g of cell lysate per lane.Perform dialysis to decrease salt concentration. Use a small dialysis device such as the Thermo Scientific Slide-A-Lyzer MINI Dialysis Device, 0.5 mL Viscous samples,(Cat. No. 88401).streaks at sample laneExcess salt (ammonium sulfate) inConcentrate and resuspend samples in lower-salt edges, dumbbell-shapedsample during gel electrophoresis buffer prior to electrophoresis. Use small-volume bands, lane widening concentrators such as Thermo Scientific Pierce Protein Concentrators PES, 0.5 mL (Cat. No. 88513).Make sure that the salt concentration does not exceed 100 mM.DNA contaminationgenomic Protein aggregationDNA in the cell lysate may cause resulting in narrowthe sample to become viscous,Shear genomic DNA to reduce viscosity before loading lanes that cannotresulting in protein aggregation,the sample.be interpreted which can affect protein migration patterns and resolutionExcess salt (sodium chloride) inPerform dialysis to decrease salt concentration. Use sample during gel electrophoresis.a small dialysis device such as the Slide-A-Lyzer MINI High salt concentrations resultDialysis Device, 0.5 mL (Cat. No. 88401).in increased conductivity, whichConcentrate and resuspend samples in lower-salt affects protein migration andbuffer prior to electrophoresis. Use small-volume can result in protein bandsconcentrators such as Pierce Protein Concentrators spreading into adjacent lanesPES, 0.5 mL (Cat. No. 88513).containing samples with normalMake sure that the salt concentration does not salt concentrations exceed 100 mM.High detergent concentrationMost of the nonionic detergents (e.g., Triton X-100, Uneven sample lanes, (e.g., SDS or Triton X-100NP-40, and Tween 20 detergents) interfere with lane widening detergent) in gel electrophoresis.SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Detergents form mixed micellesKeep the ratio of SDS to nonionic detergent at 10:1 or with the anionic detergent SDSgreater to minimize these effects.in the gel and migrate down intoUse detergent removal columns or the Thermo the gel; they interfere with theScientific Pierce SDS-PAGE Sample Prep Kit SDSprotein binding equilibrium (Cat. No. 89888) to remove excess detergent.High concentration of RIPA (radioimmunoprecipitation assay)Dilute samples before electrophoresis to lower buffer results in widening of lanesthe final concentration of lysis buffer to prevent and significant streaking duringbuffer-related defects.electrophoresisThe final concentration of reducing agents for Excess reducing agent in the lysisSDS-PAGE should be less than 50 mM for DTT Shadow at lane edges or sample buffer (dithiothreitol) and TCEP (tris(2-carboxyethyl)phosphine), and less than 2.5% for -ME (-mercaptoethanol).75'