Protein fractions were collected and concentrated using an Eppendorf Speed Vac and protein concentration determined using the Bradford assay (Bio-Rad) and PEDF ELISA (Bioproducts, MD).
Protein was concentrated (Amicon) to 5 10 mg ml−1 with protein concentration determined using a molar extinction coefficient (ϵ280nm=54890 M−1 calculatedulated with ProtParam (http://web.expasy.org/protparam).org/protparam
Membrane fractions from both rat and gerbil tissues, and CHO cells, were prepared by homogenizing in 50 mM Tris HCl buffer (pH 7.4) followed by centrifugation at 40,000 × g for 30 min. The pellet was then re-suspended in the same buffer and the protein concentration determined using the BCA reagent kit (Pierce, Rockford, IL, USA).
Aliquots of supernatant were sampled at preset intervals and the glucose concentration determined using an automated electrochemical technique (YSI 2700 Select Bioanalyser) (Yellow Springs, OH).
Immunodetection was performed using ECL plus (Amersham) and protein concentration determined using Bradford Protein Assay (BioRad, Hercules, CA, USA).
The crude lysate was cleared by centrifugation and soluble proteins concentration determined using Bradford assay (Thermo Scientific, 1856209).
The supernatant (whole cell extract) was retained, the protein concentration determined using the Bio-Rad Bradford reagent, and aliquots were stored at −80°C.
The purified DNA was resuspended in 40µl of analar water and the DNA concentration determined using a Nanodrop ND-1000 spectrophotometer.
The supernatant was collected after brief centrifugation and its protein concentration determined using the Bio-Rad DC Protein Assay Kit II (Bio-Rad, Hercules, CA).
The protein eluate was dialysed against PBS, the concentration determined using Bradford reagent (BioRad), and the solutions brought to a concentration of 50 µg/ml.
The amount of LDL is expressed in terms of protein concentration, determined using the Lowry assay with bovine serum albumin as a standard [26].
