Engineering / Regeneration Healing / Stem Cell Biology
Detection, Confirmation and Quantification of FGF2 (BOVINE)
Cambridge Healthtech Institute’s Second Annual
TISSUE ENGINEERING/REGENERATION HEALING/STEM CELL BIOLOGY
October 3-5, 1999 Pittsburgh, Pennsylvania
AGUILAR L. C., SANCHEZ J., CEJA R., SOTELO A., ALFARO F.AND ISLASA.
Instituto de Investigaciones en Neuroplasticidad y Desarrollo Celular, A.C.
Departamento de Biología Celular y Molecular, Universidad de Guadalajara.
Our group has worked out in different methods of characterization of FGF-2 molecule. Indeed, it is not a trivial task, to detect it, nevertheless the following methods are enough to detect or discard the presence of FGF2 (Bovine). First we performed the HPLC technique at 266nm (which was the optimal length wave for this molecule ), in where it was determined the Retention Time (RT) of the FGF2 (Bovine) and compared it with an FGF2 control (Sigma Chemicals, St Louis MO). After that, it was developed an ELISA test, using polyclonal antibodies against FGF-2 Bovine, developed in rabbit (R&D). It was used several proteins as negative controls (Ovoalbumin, Bovine and Human Albumin and Tripsinogen). The above mentioned proteins and the FGF-2 were fixed in the wells at 0.1, 1.0, 2.5, 5.0 and 10 µg. Finally it was done a comparative analysis between Lowry method and absorption at 280nm, in order of establish a reliable method for FGF2 quantification.
The results showed that, FGF-2 obtained in our laboratory, presented the same RT, that the FGF-2 Bovine from Sigma (RT=3.96 ± 0.06), The limit of detection of our HPLC technique was of 10 µg/ml, lower amounts were not detected in several trials.
The ELISA test showed, that the FGF-2 (from our lab) had an increase in the Optical Density data, in a dose response mode. The concentrations lower to 2.5 µg, in some experiments were not statistically different when Human Albumin was compared to our FGF-2, meanwhile amounts of 5.0, 7.5, 10 µg were significantly different (p<0.001). The differences between our FGF-2 and the other controls were all significantly, from 2.5 to 10 µg. At lower concentrations it is not possible discard the presence of FGF-2 with this method.
Finally the detection of FGF-2 and its controls, performed at 280nm, showed an excellent linearity response, meanwhile the Lowry method did not fit this requirements of linearity at the concentrations probed (10-1000µg). It is concluded, that the molecule isolated in our laboratory is indeed FGF-2 Bovine.
FGF-2 was then injected to an HPLC (Delta prep 3000 Waters (R)) with a Analytical studies were performed by HPLC with the µ Bondapak c-18 column (3.9 x 300 mm) and 80 % of TFA 0.1 % and 20% of acetonitrile as eluent, flowing at 0.8 ml/minute.Detection was performed in a spectrophotometer (Waters (R) Lambda-max mod. 481) at 266 nm; the data were integrated in a Data Module (Waters (R) 745).
Fig. 5.- FGF2 Sigma + Solvent. Monomers and Polymers
Fig. 7.- Elisa Test for FGF2 and other Proteins
Fig. 9.- Elisa Test to Confirm the FGF2 presence using Polyclonal Rabbit anti Bovine FGF2 Antibodies.
Fig. 11.- Comparative Quantification of BOVINE, OVO Albumin and FGF2 by Lowry Method
Fig. 13.- Comparative Quantification of BOVINE, OVO Albumin and FGF2 by Lowry Method