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


Introduction

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.


Methods

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.


References

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
'"/>

Source:


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:
*Name:
*Comment:
*Email:


(Date:12/8/2016)... England , December 8, 2016 ... das Unternehmen für Molekulargenetik, erweitert seine Palette an ... myPanel™ NGS Custom FH Panels, das ein schnelles ... (FH) ermöglicht. Das Panel bietet eine Erkennung von ... Variations (CNV) mit einem einzigen kleinen Panel und ...
(Date:12/8/2016)... ... December 08, 2016 , ... ... commercial launch of flexible packaging for their exceptionally efficient human mesenchymal stem/stromal ... extends RoosterBio’s portfolio of bioprocess media products engineered to radically streamline culture ...
(Date:12/8/2016)... Dec. 8, 2016 Soligenix, Inc. (OTCQB: SNGX) ... on developing and commercializing products to treat rare diseases ... that it will be hosting an Investor Webcast Event ... the origins of innate defense regulators (IDRs) as a ... oral mucositis and the recently announced and published Phase ...
(Date:12/8/2016)... to fuel Philadelphia,s innovative digital ... Southeastern Pennsylvania (" Ben Franklin "); Independence ... Cross; and Safeguard Scientifics ("Safeguard") (NYSE: SFE ... funding initiative over a four year period to grow ... burgeoning economic vitality in digital health, Ben Franklin ...
Breaking Biology Technology:
(Date:11/24/2016)... Cercacor today introduced Ember TM Sport ... non-invasively measure hemoglobin, Oxygen Content, Oxygen Saturation, Perfusion ... in approximately 30 seconds. Smaller than a smartphone, using ... to key data about their bodies to help monitor ... Hemoglobin carries oxygen to muscles. When hemoglobin ...
(Date:11/19/2016)... 18, 2016 Securus Technologies, a leading provider ... safety, investigation, corrections and monitoring, announced today that it ... to have an independent technology judge determine who has ... high tech/sophisticated telephone calling platform, and the best customer ... do most of what we do – which clearly ...
(Date:11/15/2016)... ROCKVILLE, Md. , Nov. 15, 2016 /PRNewswire/ ... clinical company developing therapeutics focused on the gut ... public offering of 25,000,000 shares of its common ... its common stock at a price to the ... gross proceeds to Synthetic Biologics from the offering, ...
Breaking Biology News(10 mins):