52 • Noreen Tuross 



PROTEIN EXTRACTS 



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stained Partially 

 Destained 



COOMASSIE 



Figure 1 . Protein extracts from three Mobridge site individ- 

 uals on an SDS 4-20% polyacrylamide gel stained with 

 Coomassie Brilliant Blue. Fully stained extracts are a smear 

 of collagen degradation products over the entire molecular 

 weight range of the gel. Equivalent extracts, partially de- 

 stained in methanol/acetic acid/water, have discrete bands 

 approximately 70 Ka and below. Multiple bands are apparent 

 in each sample. Sharp, highly stained gashes above 97 Ka 

 region are due to large amounts of protein (500 fig) applied to 

 top of each lane. 



Materials and methods 



Thirteen rib fragments, weighing from 2 to 5 g, were taken 

 from individuals (catalog numbers 325348, 325352- 

 325358, 381159, 381163, 381193, 381342 and 381345, 

 38 1 346) and extracted in 4M guanidine HC1/0.5M EDTA at 

 4°C for two days. This procedure partially deriiineralized the 

 samples. The guanidine/EDTA was removed from the sol- 

 uble protein by desalting 35 ml of the solution over a P6DG 

 column. The proteins were monitored at 254 nm, elutcd in 

 100 mM ammonium acetate, and lyophilized. 



Fractions of the desalted protein were concentrated by 

 Centricon (Amicon) filtration, and protein above 30,(X)0 mo- 

 lecular weight retained on the filter was electrophoresed. 



Mini-gradient SDS gels. 4-20% acrylamide (Novex) were 

 used in a traditional Laemmli (Laemmli 1970) buffer system. 

 The gels were stained with Coomassie Blue, and partially 

 destained for up to four days. 



Bacterial collagcnase (Advanced Biofractures) digestions 

 of up to 1 mg of protein were done at 37°C for 4 to 8 hours. 

 The fossil protein digests were then subjected to Centricon 

 filtration with a membrane that would retain all proteins 

 above 10,000 molecular weight. The filters were extracted 

 with gel sample buffer and gel electrophoresed as described 

 above. 



Electroblotting of collagenase digested proteins onto ni- 

 trocellulose was performed according to the method of Tow- 

 bin et al. (1979). Nitrocellulose electrotransfers were pro- 

 cessed for immunodetection by using 1:1000 dilution of 

 rabbit antihuman osteonectin and albumin (Cappel Laborato- 

 ries) and a 1:2000 dilution of peroxidase conjugated goat 

 antirabbit IgG (Kirkegaard and Perry Laboratories) and 

 4-chloro- l-naphthol color reagent. IgG detection was 

 achieved with a 1:1000 dilution of rabbit antihuman perox- 

 idase conjugated IgG (Capell Laboratories). 



Results and discussion 



The protein extracts from all bone samples were collagen- 

 like in their amino acid pattern. Gel electrophoresis (Figure 

 1) of the whole protein extract gave a smear of Coomassie 

 stainable material that ranged in molecular weight from 

 > 200,000 to the 30,000 retained on the Centricon filter. 

 This collagen smear is common in electrophoresed fossil 

 bone extracts (Tuross et al. 1980) and results from multiple 

 peptide bond breaks along the collagen molecule. Partial 

 destaining of these Coomassie stained gels revealed the pres- 

 ence of bands of protein originally obscured in the fully 

 stained gel (Figure 1 ). 



The fully stained fossil protein extracts and the equivalent 

 partially destained extracts are shown in relation to the pro- 

 teins extracted by guanidine/EDTA in a modem human fetal 

 calvarium. In modem bone, collagen is largely insoluble 

 when subjected to the dissociative, demineralizing condi- 

 tions of guanidine/EDTA (Termine 1983). In fossil bone, 

 however, the partial breakdown of the collagen molecule 

 renders this protein soluble to the same dissociative de- 

 mineralizing conditions. 



This increased solubility of the collagen degradation prod- 

 ucts makes the isolation and characterization of any remain- 

 ing intact noncollagenous proteins or native collagen more 

 difficult. Generally, large amounts of protein (up to 1 mg) 

 from each skeletal sample must be applied to a gel in order 

 to visualize any "bandable" protein upon partial destaining 

 of a gel. Applying a large amount of protein to a gel can lead 

 to several type of distortions, including the short, sharp, 

 gashlike disconformities seen at the top of several lines in 

 Figure I . 



Zagnb Paleopathology Symp. 1988 



