Chemistry Laboratory Classwork

Useful Stats

  • Extinction Coefficient for HICA: 43430 M-1cm-1
  • Molar Mass for HICA: 26249.96 Da
  • A(280nm)(HICA)bC(in M) (account for dilution)
  • Mol/L to mg/mL
  • Use desalting volume of protein recovered to find mg of pure protein

​Excel Data Files (dated and signed)

[[Konis&Blount 1/28/13]]


*wt ECCA Chromatograph Excel File: http://crystal-wiki.gonzaga.edu/tiki/tiki-download_file.php?fileId=163

[[Labrum, Lee, & Bolinger, 3/4/13]]


*optimized R160H HICA Chromatograph: http://crystal-wiki.gonzaga.edu/tiki/tiki-download_file.php?fileId=201
[[Labrum, Lee, & Bolinger, 5/4/13]]

Summary Results

Proof of concept work (R160H)

Protein Variant Elution Volume (mL) Elution %B (imidazole concentration) Yield in mg total cell mass (g) group member names
ECCA 34.93 mL 59% 2.84 mg 14.6 g A. Labrum, D. Lee and I. Bolinger
HICA R160H 52.70 mL 55.6% 6.7 mg 13.1 g A. Labrum, D. Lee, and I. Bolinger
HICA 42.3 mL  0% 0 mg 9.697 g M. Konis, A. Blount
HICA R160H batch gradient 100% ? ? M. Konis, A. Blount


General Method

Variant elution volume elution %B or mM imidazole yield protein (mg)  total cell pellet (g) group members
T200H/S204H HICA    127 mM imidazole     J. Birge
M194H/T196H HICA  10.3 mL 105 mM imidazole 18.3 mg 5.60 g

T. Singh, J. Nguyen

M194H/T196H HICA 90.4 mL 100 mM imidazole 13.9 mg 6.61 g J. Burr, N. Komlos
T200H/S204H HICA  9.03 mL 105 mM imidazole 0.0392 mg 5.62 g

J. Bauer, T. Heinrich

T200H/S204H HICA  12.1 mL 122 mM imidazole 10.2 mg 4.08 g H. Horney, R. Zagal


Elution volumn should be normalized to the beginning of the gradient, if possible, to account for differences in load volumes earlier in the run.