Recombinant i-fabp are subjected to electrophoresis in a 15% acrylamide gel. Protein from this gel is transferred to a pvdf transfer membrane (millipore, bedford, mass. ). The membrane is exposed to a 1:300 (v/v) dilution of rabbit anti-i-fabp and stained using an avidin-biotin system with diaminobenzidine. Western blot analysis shows a 15 kilodalton (kd) band in each lane, corresponding to the heavy band of monomeric, recombinant i-fabp. Secondary bands, representing lower molecular weight degradation products of i-fabp, are apparent in the lanes with extracts of stomach, jejunum, and ileum, but not in intestinal and liver tissue extracts. A light, 31 kd band, probably representing a protein dimer, is apparent in the lane containing recombinant i-fabp. effectiveness of daily viagra Antiserum titer. One hundred ul of the tracer (about 17,500 cpm) are incubated overnight at 4â° c. With cardinal ul of a 1:2 dilution series of rabbit anti-rat i-fabp in buffer. lowest price for generic viagra Nonspecific binding is blocked by the addition of hundred ul of normal rabbit serum (1:200, v/v). Bound antibody is separated using a second antibody method with sheep anti-rabbit immunoglobulin (igg). cheap genuine pfizer viagra Following incubation for 8 hours at room temperature, the samples are centrifuged at 2500 rpm for 15 minutes, the supernatant discarded, and the pellets counted in a gamma counter (beckman instruments, cambridge, mass. ). viagra daily bph Activity and sensitivity of the antiserum. A 2:1 (v/v) dilution series of i-fabp (1. what is viagra soft tabs 9 to 250 ng/ml) in buffer (total volume is hundred ul/sample) is incubated with c ul of rabbit anti-rat i-fabp (1:8000, v/v) and large integer ul of tracer. viagra lasts how long All samples are analyzed in duplicate. generic viagra online Following an incubation at 4â° c. , antibody is precipitated using the second antibody method. buy generic viagra The pellets are counted in a gamma counter, and a standard curve is constructed. A typical standard curve is presented in fig. 1. Ninety percent of labelled tracer (17,500 cpm/100 ul) could be precipitated at a dilution of 1:250 (v/v). Sixty percent of the label is precipitated at a titer of 18000 (v/v), and this is then used as a working dilution. Scatchard analysis reveals that the dissociation constant, kl, for this antiserum is about 3. 5ã—1010 l/mol (fig. 2). cheapest place to buy viagra online The standard curve constructed from 1. 9 to 250 ng/ml becomes linear above 3. 9 ng/ml, and this is defined as the lower limit of detection for the assay (fig. 3). I-fabp in serum, urine, and tissues. Blood and urine are obtained from 250-400 gm lbn rats (charles river, cambridge, mass. ) on a standard diet. Following ether/phenobarbital anesthesia, blood is obtained by aortic cannulation, serum from several rats is pooled, and samples are frozen at -20â° c. Urine is obtained by bladder punc. buy viagra online