Submitted to: American Leather Chemists Association Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 10/30/2006
Publication Date: 6/20/2007
Citation: Ramos, M., Latona, R.J., Marmer, W.N. 2007. Improved Monitoring of Decorin in Hides during their Processing into Leather [abstract]. American Leather Chemists Association Meeting. p. 36.
Technical Abstract: A reliable analytical method is needed to study and monitor the different interacting molecules in the processing of hides into leather. Among those molecules are decorin, biglycan, sulfated glycosaminoglycan (SGAG) and collagen. During conversion of hides into leather, some of these molecules undergo changes and removal. In a related publication from this group, ELISA was used to monitor the core protein of decorin and Alcian blue was used to estimate the SGAG content. ELISA results had indicated that almost 90% of decorin is removed from hides after dehairing and reliming; deliming and bating did not seem to remove any additional decorin. A slight anomaly was detected though where the delimed samples showed twice the amount of decorin than had been detected in the prior reliming step. Currently, we are developing an improved and more sensitive method than ELISA. Hide samples are first extracted by guanidine HCl. Dialyzing the extract removes the varying amount of salts and other ingredients and allows us to obtain a more manageable sample with uniform background and in turn more unperturbed and reliable analytical results. The high collagen content thwarted the efficient sampling for SDS-PAGE as it made the sample somewhat sticky and gave low resolution or smeary bands on the gel. To solve that problem, collagenase was added to the sample during dialysis. The dialysate is then precipitated with trichloroacetic acid (TCA) and the protein is resuspended by sonication before loading unto the SDS-PAGE gel. Changing the running buffer from Tris Glycine to Tris Tricine further improved the resolution of the protein bands in the gel. The use of a combination of two antibodies, 6D6 and PK1, gave a more sensitive and better Western blot than seen when applying either individually. ELISA results on the depletion of decorin in hide samples was evaluated and compared to Western blotting data.