b'Protein electrophoresis interfering substances Interfering substance Potential issuesSolution Excess salt High salt results in increased conductivity that Perform a sample clean-up method to lower salt causes uneven sample lanes and lane widening concentration (see Protein clean-up methods) Make sure that the salt concentration does not exceed 50100 mMDNA contamination Excess DNA causes the sample to becomeShear genomic DNA to reduce viscosity before loading viscous resulting in protein clumping, which canthe sampleresult in narrow lanes that cannot be interpretedHigh detergentDetergents can form mixed micelles with SDS Dilute the sample to reduce the final concentration of concentrationand migrate through the gel. This can lead todetergents in the samples loadedlane widening, uneven sample lanes, and streaks Remove excess detergent with detergent removal in lower regions of lanes, limiting the ability tocolumns or SDS-PAGE sample prep kitsanalyze proteins less than 40 kDaSample type andHigh protein loads can cause several issues, suchThe maximum recommended sample load for optimal protein load as loss of protein band resolution, lane streaking,resolution in mini gels with 10, 12, 15, or 17 wells is and non-straight lanes 0.5 g per band or about 1015 g of cell lysate per laneExcess/incorrectExcess reducing reagent can cause shadows atThe final concentration of reducing agents for reducing agent lane edges SDS-PAGE should be less than 50 mM for dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), and less than 2.5% for -mercaptoethanol (-ME)Excess guanidine-HCl Guanidine-HCl has high ionic strength and resultsPerform sample clean-up methodin increased conductivity that causes uneven sample lanes and lane wideningProtein clean-up methods Many detergents and salts used in protein extractionready to use and designed to eliminate potential sample formulations may have adverse effects on downstreamleakage and maximize ease of use for specific applications.analysis by protein electrophoresis. Therefore, it may be necessary to remove or reduce these contaminantsDesalting following cell lysis or subsequent sample processingSize exclusion chromatography (also known as gel such as protein purification. Listed below are thefiltration) can be effectively utilized for protein desalting. different techniques that can be used to limit theseA resin is selected with pores that are large enough interfering substances.for small contaminants (e.g., salts) to penetrate, but too small for the protein of interest to enter. This causes Dialysis the migration of small contaminants to slow as they get Dialysis is a classic separation technique that facilitatestrapped in the resin, while the larger, faster proteins the removal of small, unwanted compounds fromemerge from the column first, allowing the protein proteins in solution by selective diffusion through aof interest to be recovered separately from the small semipermeable membrane. Proteins that are larger thanmolecules retained on the column. Thermo Scientific the membrane pores are retained on the sample side ofZeba desalting products contain a unique resin and are the membrane, but low molecular weight contaminantsspecifically designed to provide consistent performance diffuse freely through the membrane and can be removedover a wide range of protein concentrations and sample over multiple buffer exchanges. Thermo Scientificsizes. High recovery of protein can be achieved even for Slide-A-Lyzer dialysis cassettes and devices aredilute protein samples.38'