SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis that is carried out in polyacrylamide gel is called polyacrylamide gel electrophoresis. Crosslinked polyacrylamide gel is formed by polymerization of monomer acrylamide in the presence of bisacrylamide. Bisacrylamide consists of two molecules of acrylamide linked by methylene group. Thus it is used as crosslinking agent. Polymerization of monomer acrylamide takes place in head to tail fashion and long chain is formed. This polymerization is an example of free radical catalysis. Polymerization is initiated by addition of ammoniumpersulfate and N, N, N’, N’-tetramethyl ethylene diamine (TEMED) and sodium dodecyl sulfate (SDS) or sodium lauryl sulfate. TEMED catalyses the decomposition of persulfate ion into free radical. This free radical is highly reactive due to the presence of unpaired electron and need to be paired with another electron to stabilize the molecule. Thus long chained polyacrylamide gel is formed. Polymerization is exothermic. Heat generated during polymerization warms gel and removes oxygen bubbles trapped in gel. Oxygen interferes with polymerization. Therefore, degassing is done before polymerization.

Polymerization is generally used in concentration between 3-30%. Pore size is determined by concentration of both acrylamide and bisacrylamide.

Acrylamide and bisacrylamide are neurotoxins. Although, polyacrylamide is not toxic, it may still contain some molecules of acrylamide and bisacrylamide that are not polymerized. Therefore, care should be taken and gloves be used to handle.

SDS is an anionic detergent which binds with most proteins in amount proportional to molecular weight of protein. One SDS molecule binds with two amino acid residues. Bound SDS contributes large amount of negative charge rendering intrinsic charge of protein insignificant and all proteins have similar charge to mass ratio. In addition, native conformation of protein is altered and opened into rod shaped structure and all proteins have similar shape. Therefore, electrophoresis in the presence of SDS is exclusively based on molecular weight of protein.

Sample to be run on SDS-PAGE is boiled for 5 minutes in sample buffer containing β-mercaptoethanol and SDS. β-mercaptoethanol reduces disulphide bridge present holding protein tertiary structure and SDS denatures and binds protein. Any protein in sample is denatured by this step with a series of negatively charged SDS along the polypeptide chain. Sample buffer also contains tracking dye bromophenol blue to monitor electrophoresis run and sucrose or glycerol that increases density allowing sample to settle down at bottom through electrophoresis buffer when injected into loading well. Once all samples are loaded, current is passed through gel. Actually, sample is not directly loaded on main separating gel but is loaded on stacking gel. Main separating gel solution is poured between glass plates and after solidification, thin layer of stacking gel is poured in which loading well is formed. Stacking gel is to concentrate protein into sharp band before entering separating gel. It is based on phenomena: isotachophoresis. Pore size in stacking gel is large and protein is stacked. Bromophenol blue (BROMOPHENOL BLUE INDICATOR 100ML) is small molecule. So it moves faster than sample protein and is used as tracking dye to monitor electrophoretic font. Once this front reaches bottom of gel, current is removed. Gel is removed and dipped in staining solution of Coomassie Brilliant Blue. Acid: Methanol in staining solution cause denatured protein to precipitate or fix and prevents from being removed from gel during washing. Destaining solution removes background dye from gel.

In SDS PAGE (Analysis of protease digestion patterns in tideland sediments using SDS-PAGE [An article from: Journal of Experimental Marine Biology and Ecology]), a single pure protein gives single band unless it is composed of two unequal subunits.

Molecular weight of protein can be determined by comparing its mobility with that of other standard protein with known molecular weight.

Rhesus monkeys in Nepal may provide new alternative for HIV/AIDS research

Scientists investigating the genetic makeup of rhesus macaque monkeys, a key species used in biomedical research have found the rhesus in Nepal may provide a suitable alternative to alleviate a critical shortage of laboratory animals used in work to develop vaccines against diseases such as HIV/AIDS.

Researchers headed by Randall Kyes of the University of Washington report that the Nepali macaques are more closely related genetically to rhesus macaques form India than rhesus macaques of China.

This is important because Indian origin animals have been used for more than half a century in biomedical and behavioral research. Rhesus macaques have contributed to the discovery of vaccines to prevent diseases such as polio and yellow fever, and represent one of the most widely used primate models for AIDS related research. India, however, banned the export of all macaques in 1978, thus leading to the current shortage. Although China has been exporting captive-bred animals for sometime, scientists have noted a number of behavioral and physiological differences in disease progression between animals from the two countries, and the Indian origin macaques are generally preferred in research on certain disease.

The new study was spearheasded by Kyes, a primatologist and head of the UW’s Division of International Programs at the Washingron National Primate Research Center, in collaboration with Mukesh Chalise, president of the Nepal Biodiversity Research Society and zoologist at Tribhuvan University in Nepal.

Blood samples from 21 Nepali rhesus macaques living at a temple site in Kathmandu were obtained as part of a comprehensive health screening conducted at the temple. These samples were compared with existing samples collected from more than 300 Indian and Chinese origin rhesus macaques.

The researchers did sequence analyses of mitochondrial DNA and examined the distribution of certain types of alleles, or pairs of genes, for genetic variations distributed across 17 chromosomes. They also looked for the presence of other alleles that are known to influence immunity and the rate of disease progression in the animals, including one that is present in Indian rhesus macaques but not in the Chinese ones.

Kyes said that the genetic analyses indicate the Nepali rhesus macaques are genetically similar to Indian origin rhesus macaques. In addition, while the temple samples were taken from an isolated population, he believes they may be representative of Nepali rhesus macaques in general given the past history of the area and the geographic barrier of the Himalayan Mountains that separates Nepal and India from China.

To facilitate the use of rhesus macaques in research while ensuring the conservation of Nepal's naturally occurring rhesus populations, the Nepali government enacted a policy in 2003 stipulating that only captive-bred animals may be used for scientific research. The rhesus macaque is prevalent in many countries and is not considered an endangered species. An agreement between the Nepal Biodiversity Research Society and the Washington National Primate Research Center was signed in September 2003 and a captive-breeding facility is now under construction. A breeding colony is expected to be established within 12 months.
Taken from www.biologynews.net (June 1, 2006)
However, due to opposition and demonstration allover the world mainly in Europe by Animal Right’s Activists, export of rhesus macaques form Nepal to USA has been halted and to my knowledge, the captive breeding facility will soon be dismantled (However, I am not sure.)