b'Preparing PAGE samples for gel loadingBefore a sample can be loaded onto a gel for analysis, it must be properly prepared. Depending on the gel type, this may involve denaturing the proteins, reducing any disulfide bonds, and adding a sample buffer. Sample buffers contain glycerol so that they are heavier than water and sink neatly to the bottom of the buffer-submerged well when loaded onto a gel. If suitable, negatively charged, low molecular weight dye is also included in the sample buffer; it will migrate at the buffer front, enabling one to monitor the progress of electrophoresis. The most common tracking dye for sample buffers is bromophenol blue. General guidelines for preparing samples are provided below.General guidelines for preparing samples for separation:Prepare your sample in the appropriate sample buffer, sometimes referred to as loading buffer, such that the final concentration of the sample buffer is 1X. Recommended sample buffers are listed on page 42.Running reduced and nonreduced samples: For optimal results, we do not recommend running reduced and nonreduced samples on the same gel. If you do choose to run reduced and nonreduced samples on the same gel, do not run reduced and nonreduced samples in adjacent lanes. The reducing agent may have a carry-over effect on the nonreduced samples if they are in close proximity. Heating samples: Heating the sample at 100C in SDS-containing buffer results in proteolysis [3]. We recommend heating samples for denaturing electrophoresis (reduced or nonreduced) at 70C for 210 minutes for optimal results. Do not heat the samples for nondenaturing (native) electrophoresis or Invitrogen Novex Zymogram Plus gels.41'