Navigation Links
Fractionation of Bovicola ovis Homogenates Using the Mini Whole Gel Eluter

Brian J. Shiell and Wojtek P. Michalski, CSIRO Division of Animal Health, Australian Animal Health Laboratory, Geelong, Victoria, Australia


Amino acid sequence and composition analyses of proteins can be performed on samples derived from polyacrylamide gels. Protein electro-elution, the most commonly used method of recovering proteins from gels, can be performed on cut-out gel slices or using the Whole Gel Eluter recently introduced by Bio-Rad.14 The Mini Whole Gel Eluter was used here to fractionate crude homogenate of the common sheep louse with the aim of characterizing immunogenic components.


Common sheep lice, Bovicola ovis, were harvested from infested animals, homogenized by ultrasonication, and the homogenate was clarified by centrifugation on a benchtop microfuge. Homogenate samples (1 mg protein) were separated by SDS-PAGE in 12% preparative well Tris-HCl Ready Gels (Bio-Rad). Protein fractions were immediately electro-eluted at 100 mA for 25 min using the Mini-Whole Gel Eluter. Three elution buffers: (A) 100 mM NH4HCO3, 0.01% SDS, pH 8.5, (B) 10 mM phosphate buffer, 0.005% SDS, pH 7.8, and (C) 25 mM Tris, 19.2 mM glycine, 0.01% SDS, pH 8.3, were used to determine the elution efficacy. The fractions were analyzed in 12%, 15 well Tris-HCl Ready Gels (Bio-Rad) and proteins were visualized by Coomassie blue staining. Bio-Rads low molecular weight standards for SDS-PAGE are shown in Figure 1B, lane b.

Results and Discussion

Louse homogenate preparations are often rich in lipids and do not separate well on SDS-PAGE gels ( Figure 1A, lane a). Individual proteins are poorly resolved and their extraction from individual cut-out bands is often inefficient. Several attempts were made to obtain amino acid sequence from various protein bands of louse homogenate using a variety of standard methods. This approach proved to be relatively ineffective and time-consuming.

The Mini Whole Gel Eluter was then used to fractionate crude louse homogenate. Samples of louse homogenate were separated on 12% preparative well gels and subjected to electro-elution. Two elution buffers, B and C, varying slightly in pH (7.8 v. 8.3) and in SDS content (0.005% v. 0.01%), were applied and in both experiments elution resulted in good recovery of a majority of proteins. It should be noted, however, that high molecular proteins did not elute well in buffer (B) containing low concentration of SDS (0.005%). The application of NH4HCO3/SDS buffer (A) resulted in virtually no protein elution, possibly due to the low stability of ammonium carbonate buffer.

It was obvious that the buffer (C) containing Tris and a higher SDS concentration (0.01%) was a much better choice, as it resulted in the elution of virtually all proteins present in the louse homogenate ranging from <14,000 to >100,000 daltons. Increased SDS concentration also yielded approximately 23 fold greater amount of protein eluted in each of 14 fractions (Figure 1B).

Protein fractions electro-eluted on the Mini Whole Gel Eluter with buffer C, containing 0.01% SDS, pH 8.3, were transferred on Prosorb, desalted, and subjected to automated N-terminal amino acid sequencing. Of three major proteins analyzed, sequence information was obtain ed for two. One of the proteins contained a sequence that was similar to that of a reported allergen.


1. Andersen, P. and Heron, I., J. Immunol. Meth., 161, 29 (1993).

2. Dunbar, B. and Wilson, S. B., Anal. Biochem., 216, 227 (1994).

3. Andersen, P., Andersen, . B., Srensen, A. L. and Nagai S., J. Immunol., 154, 3359 (1995).

4. Brandt, L., T. Oettinger, A., Holm, Andersen, . B. and P. Andersen, J. Immunol., 157, 3527 (1996).

Coomasie is a trademark of ICI.

back to top


Page: All 1 2 3

Related biology technology :

1. Efficient Cellular Fractionation for RNA and Protein Isolation
2. Sample Preparation Solutions for Cellular Protein Fractionation Poster, Rev A
3. Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
4. Oil-Free PCR Using the Hot Top Assembly
5. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
6. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
7. Using XL1-Red Mutator Strain to Generate Esterase Variants
8. Detect Released Oligosaccharides Using the Eagle Eye II Still Video Imaging System
9. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
10. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
11. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
Post Your Comments:

(Date:6/27/2016)... DIEGO , June 27, 2016  Sequenom, Inc. ... committed to enabling healthier lives through the development of ... Court of the United States ... courts that the claims of Sequenom,s U.S. Patent No. ... patent eligibility criteria established by the Supreme Court,s Mayo ...
(Date:6/27/2016)... (PRWEB) , ... June 27, 2016 , ... Parallel ... clinical trials, announced today the Clinical Reach Virtual Patient Encounter CONSULT module ... circle with the physician and clinical trial team. , Using the CONSULT module, patients ...
(Date:6/27/2016)... (PRWEB) , ... June 27, 2016 , ... ... operations for Amgen, will join the faculty of the University of North ... adjunct professor of strategy and entrepreneurship at UNC Kenan-Flagler, with a focus on ...
(Date:6/27/2016)... June 27, 2016   Ginkgo Bioworks , a ... engineering, was today awarded as one of the ... the world,s most innovative companies. Ginkgo Bioworks is ... the real world in the nutrition, health and ... directly with customers including Fortune 500 companies to ...
Breaking Biology Technology:
(Date:6/9/2016)... 2016 Paris Police Prefecture ... security solution to ensure the safety of people and operations ... the major tournament Teleste, an international technology group ... announced today that its video security solution will be utilised ... up public safety across the country. The system roll-out is ...
(Date:6/2/2016)... The Department of Transport Management (DOTM) ... million US Dollar project, for the , Supply ... Enrolment, and IT Infrastructure , to ... implementation of Identity Management Solutions. Numerous renowned international vendors participated ... was selected for the most compliant and innovative ...
(Date:6/1/2016)... June 1, 2016 Favorable Government ... Administration and Criminal Identification to Boost Global Biometrics System ... released TechSci Research report, " Global Biometrics Market ... Forecast and Opportunities, 2011 - 2021", the global biometrics ... 2021, on account of growing security concerns across various ...
Breaking Biology News(10 mins):