b'Protein extraction and sample preparation for electrophoresisGood sample preparation is critical for successfulcompatible with the buffer ones sample is prepared in separation of protein bands in electrophoresis. No singleis the simplest route. However, if one cannot change sample preparation method or buffer will work for allthe gel electrophoresis chemistry system, one may sample types due to the diversity of protein samples.need to perform sample clean-up to render the sample However, the following general guidelines below shouldcompatible with the given system (see Protein clean-up help when preparing samples for protein electrophoresis.methods section). Certain clean-up methods are more favorable than others for reducing or removing specificTo minimize sample variability, keep sample preparationinterfering substances. workflows simple, and use reagents optimized for the specific sample type and target proteins. To avoid under- or overloading samples, determine the protein concentration of each sample prior toCell lysis disrupts cell membranes and organelles,electrophoresis with a compatible protein assay resulting in unregulated enzymatic activity that can(see Protein quantitation section). Note that certain reduce protein yield and lead to degraded proteins.buffer components can interfere with the chemistry To prevent these negative effects, protease andof a given protein assay, and these components may phosphatase inhibitors should be added to theneed to be reduced or removed from the sample lysis reagents. prior to determining sample concentration. TheseSome buffer components may interfere with the choseninterfering substances can cause artificially high or low gel electrophoresis chemistry system (e.g., Tris-glycine,concentration readings, depending on the substance and Bis-Tris) and cause a variety of artifacts when runningthe given protein assay. the gel. Selecting a gel electrophoresis chemistry that is Protein sample preparation for electrophoresisStep 1 Step 2 Step 3Cellular disruption and protein extraction Contaminant removal,Quantitation Different samples (e.g., plant vs. mammalian cells)desalting, concentration Quantitate protein samples using will require different disruption and extractionRemove or reduce interferinga compatible protein assay. strategies. Use reagents that have been optimizedsubstances that can negativelyConcentrate or dilute samples for specific sample types. In addition, use proteaseimpact electrophoresis.if necessary.and phosphatase inhibitors to minimize activity of proteases that can modify and reduce protein yields. For long-term storage of samples, we recommend freezing them to further inhibit protein degradation. 36'