Kit for fast, easy concentration and purification of samples for SDS-PAGE
• Purify - Remove contaminants that can interfere with electrophoresis (GuHCl, urea, Ammonium sulfate, etc)
• Quick - Protein samples ready to load in less than 10 minutes
• Safe - and non-toxic - no DMSO required
• Compatible with SDS-PAGE and downstream Western blotting
• Many times faster than concentration and buffer exchange via ultrafiltration or dialysis
Figure 1: Afyon protocol compared to traditional purification protocol.
Afyon concentration is compatible with Western blot detection.
Figure 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). The blots were detected using the WesternBright MCF multicolor fluorescent Western blotting kit. No bands were detectable in the diluted cell extract when stained for either GAPDH (panel a, lane 3) or SRC (panel b, lane 3). However, after Afyon concentration, bands are easily visualized for both proteins (lanes 4). Band intensities are indicated.
Figure 3:Afyon removes buffer components such as guanidinium chloride, thiocyanate or urea that can cause samples to migrate irregularly. A sample of K-561 cell lysate in 4 M guanidinium thiocyanate spreads and runs irregularly on a gel (lane 2). When the protein is recovered using Afyon resin (lanes 4-5), the salt is removed and the sample runs cleanly. 10 μl of Afyon recovers 2.5 μg protein (lane 4), while 20 μl of Afyon recovers 5 μg protein (lane 5). MW = molecular weight markers.
Figure 4:. HeLa cell lysate was diluted to 20 μg/ml with 1M NaCL, 20 mM Tris pH 7.6. 1 ml diluted lysate was concentrated and buffer exchanged using an ultrafiltration spin filter (Milipore), or Afyon resin. Ultrafiltration took over one hour, while Afyon took less than 10 minutes. Lane 1: molecular weight markers. Lane 2: sample concentrated by ultrafiltration. Lane 3: sample concentrated using Afyon.