Navigation Links
Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C

Dr Albert J Ketterman and SHP Parakrama Karunaratne, Dept. of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT UK


In attempts to control Culex quinquefasciatus as a worldwide vector of diseases such and filariasis and encephalitis, mosquito field populations are sprayed with organophosphate insecticides. However, mosquitoes develop tolerance to organophosphate.

One of the mechanisms of organophosphate resistance involves either a different form and/or increased amounts in a carboxyolesterase (EC Carboxylesterase A2 is one of two enzymes found elevated together in Culex mosquitoes. The putative mechanism involving the resistance-conferring carboxylesterase A2 is an increased expression in this enzyme for the sequestration of, and cross-resistance to, various organophosphate and carbamate insecticides (Raymond et al. 1991).

The question of a qualitative change in the carboxylesterase has not been fully explored with a purified enzyme. Here we report the use of Bio-Rads Model 491 prep cell for preparative, nondenaturing gel electrophoresis as a final step in purifying the native carboxylesterase A2.

Carboxylesterase A2 was isolated from the supernatant of a mosquito homogenate by sequential ion exchange, hydrophobic interaction, and hydroxyapatite chromatography, followed by a final purification step using the Model 491 prep cell (Ketterman et al. 1992).

Preparative Native PAGE
As suggested in the Model 491 prep cell instruction manual, the gel composition (monomer concentration) for maximum resoluti on was determined empirically by analytical, nondenaturing PAGE using the Mini-PROTEAN II cell (Figure 1).

Native PAGE separates proteins both by charge and size and we had protein bands above and below our A2 protein band of interest. At each gel concentration used, the distance was measured between the band of interest and the nearest bands above and below it. The gel concentration was 9%T/3%C where the two curves intersect (Figure 1). The electrode running buffer, in both the upper and the lower buffer chambers, and the buffer used to prepare the gel, was 0.1 M Tris-borate (pH 8.0) containing 2.0 mM EDTA. The sample, after elution from hydroxyapatite, was concentrated using a Centriprep 10 concentrator (Amicon) to approximately 0.5 ml. The sample remained in the elution buffer from the hydroxyapatite column, that is, approximately 60 mM phosphate buffer (pH 6.8). The concentrated sample was mixed with a 5x solution of glycerol and marker dye Xylene Cyanole FF to give a final concentration of 10% glycerol and 0.02% dye marker. The sample, approximately 1.6 mg of protein, was loaded on a 9%T/3%C, 6 cm gel in the Model 491 prep cell (28 mm ID). The Model 491 prep cell had previously been equilibrated overnight in a 4C coldroom. Electrophoresis was performed at 15 W constant power (350400 V). Elution (1 ml/min) and fraction collection (5 ml fractions) began when the marker dye eluted from the gel, after about 1.5 hr run time (Figure 2). The enzyme activity peak was completely eluted about 3.75 hr later. The total run time was 5.5 hr. Electrode buffer was also used as elution buffer because the enzyme is stable in this buffer.

Results As shown in Table 1, there was an 87.0% recovery of enzyme activity applied to the Model 491 prep cell. The protein appeared to be homogeneous as determined by SDS-PAGE (Figure 3). In addition, several other esterases that appear as electrophoretic allozymes on native PAGE also have been separated by the Model 491 prep cell under the conditions described above. The purified enzymes have been characterized (by kinetics) with various insecticides to determine that qualitative differences exist in mosquito populations from different geographic locations.

Ketterman AJ et al., Purification and characterization of a carboxylesterase involved in insecticide resistance from the mosquito Culex quinquefasciatus, Biochem J 287, 355360 (1992)

Raymond M et al., Worldwide migration of amplified insecticide resistance genes in mosquitoes, Nature 350, 151153 (1991)

back to top


Page: All 1 2 3

Related biology technology :

1. A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF
2. Detection of Platinum Species in Plant Material by Preparative Isotachophoresis Using the Model 491 Prep Cell, Rev A
3. Preparative Nondenaturing Gel Electrophoresis to Purify NADP-Specific Glutamate Dehydrogenase From Chlorella, Rev B
4. Preparative SDS-PAGE Electrophoresis of a Recombinant Epstein-Barr Virus Encoded Protein and Its Application in Serodiagnostic Test Systems, Rev C
5. Purification and Characterization of beta-Lactoglobulin Genetic Variants A and B Using Preparative Electrophoresis and Isoelectric Focusing
6. A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF
7. Semi Preparative Purification of Proteins
8. A Versatile Power Supply for All Electrophoresis Applications
9. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
10. Continuous-Elution Electrophoresis for Purification of the Baculovirus-Expressed Coronavirus Structural Proteins, Rev A
11. Detection of K-ras Point Mutations in the Pancreas by Constant Denaturing Gel Electrophoresis Using the DCode System
Post Your Comments:

(Date:10/8/2015)... 2015  Genetic testing for mutations beyond those currently ... more couples at risk of having a child with ... the 2015 American Society of Human Genetics (ASHG) Annual ... Baltimore, Maryland . The study is one of ... ) are presenting at the conference this week demonstrating ...
(Date:10/8/2015)... Oct. 8, 2015 Celemics, a global pioneer ... the North American market with the establishment of Celemics ... and Europe , has secured itself ... Enrichment methodology and sample preparation for DNA sequencing and ... hereditary and somatic genetic testing more accurately, quickly, and ...
(Date:10/8/2015)... 2015 Nobel Prize in Chemistry has been awarded to ... Aziz Sancar , who earned his PhD in molecular and cell ... one of three scientists who received the Nobel Prize for their ... a molecular level.  --> Aziz Sancar , who earned ... Dallas in 1977, is one of three scientists who ...
(Date:10/8/2015)... 8, 2015 /PRNewswire/ - RepliCel Life Sciences Inc. (OTCQB: REPCF) ... on the development of autologous cell therapies, today announced its ... taking place in the month of October: ... RepliCel will be presenting at the annual Partnering ... Mesa to be held October 7-9 in ...
Breaking Biology Technology:
... was at a security conference and stayed at a hotel, ... claimed that they had implemented it so only those on ... ,I do not purchase wireless Internet service when Im at ... the seminar hosts office. Out of curiosity, on my second ...
... Commerce has announced that it is producing the Wisconsin ... Thursday, September 25, from 8:30 a.m. to 3:45 p.m. ... , ,Wisconsins medical device and technology cluster is ... state, said Department of Commerce (Commerce) Secretary Cory L. ...
... all things e-commerce will speak to cutting-edge Madisonians Thursday evening ... Club Majestic, 115 King Street in downtown Madison. , ,Nationally ... about how e-commerce is coming out of the weeds after ... Some companies are finally learning how to please consumers and ...
Cached Biology Technology:
(Date:9/10/2015)... LONDON , Sept. 10, 2015 Report ... thus far not quite delivered upon previous expectations of ... might be the breakthrough year in which wearables begin ... been expected. One of the main reasons is the ... disrupt not only the SmartWatches market, but the overall ...
(Date:9/8/2015)... , Sept. 8, 2015   TREW Marketing ... new survey, Smart Marketing for Engineers TM : ... engineering professionals reveals insightful findings about engineers, preferences for ... of content engineers most value and trust, and where ... Marketers targeting technical audiences grapple with how to best ...
(Date:9/2/2015)... Biometric technology ... system which uses identical human characteristic such as ... and finger print. Increasing number of terror attacks ... which provides high level of security. Traditional security ... are vulnerable to attacks. Furthermore, Biometric technology uses ...
Breaking Biology News(10 mins):
... adults living closer to a fast food restaurant had a ... away from fast food, according to researchers at The University ... particularly strong among those with a lower income. ... of Public Health indicates higher BMI associates with residential ...
... relationship between the heritable risk for schizophrenia and low ... associated with cognitive impairments that may cause functional disability. ... to the risk for developing schizophrenia. For example, reduced ... Also, these deficits may be present in healthy relatives ...
... hematopoiesis at sites outside the bone marrow in ... (LMU) in Munich now show that a specific type ... blood cells. Balanced hematopoiesis is essential for the ... takes place mainly in the liver and the spleen. ...
Cached Biology News:
... Blunt TOPO PCR Cloning ... designed for cloning and ... PCR products. The pCR4Blunt-TOPO ... Cloning and greater than ...
... 5 ml CELLection Dynabeads (4.5 m) coated ... epithelial antigen EpCAM and DNase Releasing Buffer. ... of epithelial tumour cells directly from blood, ... can be enriched from bone marrow. Enriched ...
Recombinant Human CCL21/6Ckine (Mucin Stalk Chimera)...
Recombinant Human Erythropoietin (Tissue Culture Grade)...
Biology Products: