HPLC Analysis of SigmaMAb Monoclonal Antibody Subunits on BIOshell™ A400 Protein C4

HPLC Analysis of SigmaMAb Monoclonal Antibody Subunits on BIOshell™ A400 Protein C4

Conditions

column BIOshell A400 Protein C4, 5 cm X 2.1 mm I.D., 3.4 µm particles (66824-U)
column temp. 75 C
mobile phase [A] 0.1% TFA in water:0.1% TFA in acetonitrile (80:20, v/v); [B] 0.1% TFA in water:0.1% TFA in acetonitrile (40:60, v/v)
gradient 30 to 42% B in 5 min
flow rate 0.3 ml/min
pressure 720 psi (initial)
injection 1 µl
detector UV, 215 nm

Description

Analysis Note Complete reduction of IgG can dissociate the protein into its two component polypeptides: light chain (~ 25 kDa) and heavy chain (~ 50 kDa); the reaction can be enhanced in the presence of a denaturant, such as 8 M guanidine-HCl. Resolution of the denatured, reduced subunits is readily achieved by reversed-phase chromatography on BIOshell A400 Protein C4 column. Dithiobutylamine (DTBA; p/n 774405) was used because it is a more reactive than the alternative dithiol DTT; DTBA and DTT have similar reduction potentials, but the pKa for DTBA is a pH unit lower than that of DTT.
Categories Analytical Chromatography, Proteins, Peptides
Featured Industry Life Science and Biopharma
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
Other Notes SigmaMAb is dissolved in 0.1 M ammonium bicarbonate, 8 M guanidine-HCl, 0.1 M dithiobutylamine; final conc. = 5 g/L. At 1 h, an aliquot to be chromatographed, is removed and diluted 25x with water
suitability application for HPLC

Materials

     
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