1 ml diluted lysate was concentrated and buffer exchanged using an ultrafiltration spin filter (Milipore), or Afyon resin. HeLa cell lysate was diluted to 20 μg/ml with 1M NaCL, 20 mM Tris pH 7.6. MW = molecular weight markers.Ĭoncentrate samples 10 times faster than spin ultrafiltration.įigure 4. 10 μl of Afyon recovers 2.5 μg protein (lane 4), while 20 μl of Afyon recovers 5 μg protein (lane 5). When the protein is recovered using Afyon resin (lanes 4-5), the salt is removed and the sample runs cleanly. A sample of K-561 cell lysate in 4 M guanidinium thiocyanate spreads and runs irregularly on a gel (lane 2). Band intensities are indicated.Īfyon removes contaminants that can interfere with electrophoresis.įigure 3:Afyon removes buffer components such as guanidinium chloride, thiocyanate or urea that can cause samples to migrate irregularly. However, after Afyon concentration, bands are easily visualized for both proteins (lanes 4). No bands were detectable in the diluted cell extract when stained for either GAPDH (panel a, lane 3) or SRC (panel b, lane 3). The blots were detected using the WesternBright MCF multicolor fluorescent Western blotting kit. Prepare samples in as little as 10 minutes.įigure 1: Afyon protocol compared to traditional purification protocol.Īfyon concentration is compatible with Western blot detection.įigure 2: Western blots were created with samples of A431 cell extract (panels a and b, lane 2), diluted cell extract (lane 3) and the diluted extract concentrated using the Afyon protocol (lane 4). Purification successfully removes comtaminants that can interfere with electrophoresis (Figure 3) an shows similar results to ultracentrifugation while being ten times faster (Figure 4). Samples remain compatible with both chemiluminescent and fluorescent Western blotting (Figure 2). Additionally, the Afyon protocol is easily scaled up, allowing multiple samples to be prepared in parallel. The fast, efficient protocol generates samples ready to load on a gel in less than ten minutes (Figure 1), much more quickly than can be achieved with alternate methods such as dialysis, acetone or TCA precipitation. The Afyon SDS-PAGE sample preparation kit provides a means to quickly concentrate protein samples, and separate them from buffers that interfere with electrophoresis. Concentrate and purify protein samples for SDS-PAGE with the Afyon sample purification kit
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