Beruflich Dokumente
Kultur Dokumente
By Sreedharan Lakshminarayanan
outline
Breast cancer
Statistics
Risk factors Types of Breast cancer
Western Blotting
Breast cancer
Cancerous growth in breast tissue
Multiply uncontrollably
The cause is unclear Several risk factors
Age
Genetic Family history
Menstrual periods
Breast radiation early in life Alcohol
Smoking
Lack of exercise
Statistics
1.5 million worldwide.
http://www.komennyc.org/site/PageServer?pagename=breasthealt h_statistics
recognition and destruction. HLA can act as biomarker but it cannot convey information. Class I MHC responsible for conveying the intracellular to immune system. Then peptides are recognized by cytotoxic T lymphocytes.
7) and non cancer cell line (MCF 10 A) are cultured in a suitable medium. sHLA obtained by deletion of transmembrane and cytoplasmic domain. sHLA and breast cell line and non cancer cell line are mutated for a certain period.
ELISA
As the name describes enzyme-linked
immunosorbent assay, where the reaction of antigen and antibody takes place in vitro and those reactions are monitored by enzyme measurements. Ninety-six microwell plates is used. Antibody is immobilized on each well of micro plate
Peptide separation
Purified peptide
Detected by UV absorption
Mass Spectroscopy
Peptides are Ionized by elecrospray
Western Blotting
Also known as immunoblotting or protein blotting. Used to confirm the protein expression. Steps involved in western blotting:- Sample preparation - Electrophoresis - Transfer of proteins and staining
Sample preparation
Tissue are lysed to release protein
Maintained in a buffer
Centrifuged Proteins were used for further separation.
Gel electrophoresis
- Polyacrylamide gel electrophoresis are being
used in this experiment - Proteins are separated according to the charge in first dimension and according to the mass in second dimension on gel electrophoresis by applying electrical current. - Separated proteins are transferred or blotted.
from gel to a sheet of blotting paper called nitrocellulose. The proteins will move out from the gel onto the surface of the nitrocellulose membrane. Copy of protein pattern will be on the membrane. Dyes are added to the gel to confirm the transfer of protein. Membrane is incubated with an specific antibody to the particular protein.
Continuous.
Then antibody coupled with enzyme and
incubated with the substrate. After reaction with enzyme, luminescent will be produced which is used for the identification.
http://www.virology.ws/2010/07/07/virology-toolbox-the-western-blot/
Comparison
ELISA gives a +ve or ve test result based on
the antibody. Whereas western blotting is more specific and it allows to visualize the antibodies against the viral protein.
References
O. E. Hawkins, R. S. VanGundy., Identification of Breast Cancer
Peptide Epitopes Presented by HLA-A*0201 Journal of Proteome Research 2008, 7, 14451457. J.P. Carralot, C. Lemmel, Mass spectrometric identification of an HLA-A*0201epitope from Plasmodium falciparum MSP-1., International Immunology, 2008 Vol. 20, No. 11, pp. 14511456. ONeil, K. A.; Miller, F. R.; Barder, T. J.; Lubman, D. M. Profiling the progression of cancer: separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis. Proteomics 2003, 3 (7), 125669.