Manufacturers use HPLC-UV or electrophoresis methods to determine protein/peptide purities, which are unable to detect nonresponsive contaminations (e.g., dried buffer salts) and thus may result in a positive bias.
After hybridization, slides were washed using commercial Agilent Wash Buffer and Stabilization and Drying Buffer (Agilent Technologies, Santa Clara, CA) and scanned using GenePix 4100A scanner (Molecular Devices Inc., Sunnyvale, CA) Median signal intensities for each spot were background-corrected and log-transformed.
Gelatin crosslinking (10 and 40 mM glutaraldehyde), BMP-2 dose (6 and 60 ng BMP-2 per mg dry microparticles), buffer type (phosphate buffered saline (PBS) and collagenase-containing PBS), and gelatin type (acidic and basic) were investigated for their effects on BMP-2 release.
Masses were obtained by filtering solutions in equilibrium coupled to drying in buffer at 60 °C for 24 h.
The maxisorp plates (Nunc) were coated overnight with 15 µM BCCP in phosphate buffered saline (PBS) at 4°C.The wells were washed with PBS and dried off buffer.
Gels were washed twice in running buffer, dried (Gel Drying System 583, Bio-Rad Laboratories, München, Germany) and subjected to autoradiography.
The membranes were immersed at room temperature in phosphate-buffered saline supplemented with 0.2% Tween 20 and 5% nonfat dry milk (blocking buffer) for 1 h before incubation with primary serum (diluted 1 1,000 in blocking buffer) for 1 h.
Membranes were blocked with TBS or PBS (pH 7.4) containing 0.1% Tween 20 and 5% nonfat dry milk (blocking buffer), and incubated overnight with primary antibodies in the same buffer.
Nonspecific protein binding on the membranes was blocked using Tris-buffered saline with 0.05% Tween 20 (TBST) and 10% non-fat dry milk (blocking buffer).
The swelling ratio (SR) of the hydrogels was determined by immersing the dry hydrogels in buffer solutions (prepared from Na2HPO4 and NaH2PO4, pH = 7.2 7.4) at room temperature.
