Page 341 - Fundamentals of Light Microscopy and Electronic Imaging

324 APPENDIX II

Notes on Preparing Stock Solution of Serum Albumin
1. Dialyze 50 mL of 30% (wt/vol) bovine serum albumin (Sigma liquid stock A
7284) against 2 L distilled water to remove 158 mM NaCl. Change water after
1–2 hr, then continue overnight with 2 L water containing 30 mL ion exchange
resin (BioRad AG 501-X8 mixed-bed resin) on a stirring box at 4°C.
2. Transfer the albumin to a lyophilizer jar, freeze in liquid nitrogen or dry
ice/ethanol bath with swirling, and lyophilize overnight until dry.
3. Resuspend the dry albumin powder in 25 mL distilled water. Break up clumps of
albumin periodically during the day. It takes several hours to resuspend the albu-
min. The solution may need to be centrifuged to remove bubbles and foam using
a benchtop centrifuge.
4. Determine the protein concentration by reading absorbance at 280 nm in a UV
spectrophotometer. Dilute the stock 1:1000 in water before reading. Use care in
performing the dilution, since the BSA stock is extremely thick and concen-
trated. The OD 280 of a 1 mg/mL solution is 0.66. The concentration of the stock
will be ≥ 400 mM.
5. Prepare the following 5 solutions as indicated. The final total solute concentra-
tion in each tube will be 326 mOsM. All solutions contain added sodium chlo-
ride to maintain the erythrocytes. NaCl is added to give the same value of
osmolarity for each tube, but albumin contributes 95–98% of the refractive
index, the balance being due to the salt, which we will ignore. (If albumin is pre-
pared at other concentrations, the following values will be useful: from tables,
the osmolarity equivalent of 100 mg/mL BSA 13.793 mM NaCl, and 9.463
g/L NaCl 326 mOsM.)

mg/mL BSA g NaCl/L
200 7.863
250 7.463
300 7.063
350 6.663
400 6.263

References: Barer et al., 1953; James and Dessens, 1962.

Chapter 8: Polarized Light

Materials

2 dichroic sheet polarizers
light box or overhead projector
microscope slide
sheet of cellophane (not food wrap)
calcite crystals