Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel.
To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.
Casas-Terradellas
Eduard
E
Departament de Ciències Fisiològiques II, IDIBELL, Campus de Bellvitge, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.
Garcia-Gonzalo
Francesc R
FR
Hadjebi
Ouadah
O
Bartrons
Ramon
R
Ventura
Francesc
F
Rosa
Jose Luis
JL
D000178:Acrylamides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000302:isolation & purification; D020662:Guanine Nucleotide Exchange Factors / Q000302:isolation & purification; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D044767:Ubiquitin-Protein Ligases
D000178:Acrylamides; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C102169:HERC1 protein, human; D044767:Ubiquitin-Protein Ligases