A laboratory exercise for visible gel filtration chromatography using fluorescent proteins.
Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified, and used in a laboratory exercise to intuitively demonstrate GFC. Different bands, corresponding to RFP, RFP-CFP (RC), YFP-RFP-YFP (YRY), and pyruvate kinase II-GFP (PKG) were well separated on a Superdex 200 column from a 0.5-mL sample. Increasing the sample volume and changing the chromatographic resin to Sephadex G-100 resulted in lower resolution separation. Students enjoyed identifying combinations of colored proteins and found this exercise helpful for understanding the factors that affect GFC resolution.
Department of Biology, College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, China, 321004.
Wenqiang
W
Zhang
Yibin
Y
Cao
Lishan
L
Xu
Jufang
J
Gong
Meihao
M
Sun
D002850:Chromatography, Gel / Q000379:methods*; D003911:Dextrans / Q000737:chemistry; D004493:Education; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007753:Laboratories; D008164:Luminescent Proteins / Q000737:chemistry; D000097573:Red Fluorescent Protein
D003911:Dextrans; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein; C025614:sephadex
fluorescent protein; gel filtration chromatography; laboratory exercise