Cameron's LaBLog

 

On the basis of a 25 mg/mL solution, assuming a density of the protein of 1.3, we get an excluded volume of 19uL per mL or 1.9% of the volume.

The molecular formula for lysozyme (calculated at ExPASy with ProtParam program) is C705H1116N214O204S12 (2251 atoms total, MW is 16238.6)

So as to use the same numbers as have been calculated for the buffers we will just assume 2% as our densities and concentrations are not accurate enough to justify higher precision.

So for each atom I need 2% of the molar atomic composition of the protein as follows:

Atom	mols in 1 mol lys	molar proportion	2% amount(25 mg/mL)	10mg/mL	5 mg/mL	2 mg/mL
C	705	0.313	0.00626	0.00250	0.00125	5E-4
H	1116	0.496	0.00992	0.003968	0.00199	7.9E-4
N	214	0.0951	0.00190	0.00076	3.8E-4	1.52E-4
O	204	0.0906	0.00181	0.000724	3.62E-4	1.5E-4
S	12	0.00533	0.000107	0.0000428	2.14E-5	8.6E-6

Project: NIMROD-March-2012
Lysozyme (8 x ~0.5g) was weighed out and dissolved in 5-10 mL of each of the buffers prepared in Buffer Prep for NIMROD Experiment. Each sample was dialysed against its buffer at least 3 x 200 mL. A mistake was made in preparing the H20 20 K sample and so this buffer was re-made and the sample dialysed a further 3 x 200 mL in the new buffer. Similarly a problem with the D2O 200 K buffer meant that further dialysis was required to be sure that the buffer was correct.

Samples were then centrifuged and supernatant poured off to give:
Lysozyme in H2O 20 mM KCl
Lysozyme in H2O 200 mM KCl
Lysozyme in H20 20 mM NaCl
Lysozyme in H2O 200 mM NaCl
Lysozyme in D2O 20 mM KCl
Lysozyme in D2O 200 mM KCl
Lysozyme in D2O 20 mM NaCl
Lysozyme in D2O 200 mM NaCl

Procedure: UV-Vis
Each sample was diluted 5 uL plus 495 uL of buffer and the UV-Vis Spectrum obtained.

Sample	Data
Lysozyme in H2O 20 mM KCl
Lysozyme in H2O 200 mM KCl
Lysozyme in H20 20 mM NaCl
Lysozyme in H2O 200 mM NaCl
Lysozyme in D2O 20 mM KCl
Lysozyme in D2O 200 mM KCl
Lysozyme in D2O 20 mM NaCl
Lysozyme in D2O 200 mM NaCl

Measured concentration 50.4 mg/mL

Required for 2 mL of 25 mg/mL: 0.992 mL

Measured concentration 49.1 mg/mL

For 2mL of 25 mg/mL require 1.018 mL