Flashcards in Test 2, 15. analysis of cells, molecules, and systems Deck (38):
What is a cell culture?
1. removing cell form organism and promoting their growth in a favorable artificial envrionment
What is a primary cell culture?
1. derived from animal directly
2. survive for finite time period
3. mechanical tissue disruption and isolation of tissue and cells to obtain sample
What is a continuous cell line?
1. line of primary cells that have become immortal bc of transformation
2. tumor derived, or from viral transformation (epstein barr)
Primary cell cultures are finite, and can only divide a number of times. This is senescence or________?
1. the loss of the ability to proliferate, based on genetic
What are characteristics of cell line cultures>
1. more differentiated phenotype
2. homogenous population
3. infinite in vitro life span
4. immortalized from spontaneous genetic mutation
What are some examples of cell line cultures?
2. parkinson's model of disease
What morphological categories are cell lines divided into?
2. epithelial like cells
3. lymphoblast like cells
What are fibroblastic cells?
1. mammalian cell line
3. elongated shape that grows onto substrate
What are epithelial-like cells?
1.mammalian cell line
2. polygonal shape
3. attach to substrate in discrete patches
What are lymphoblast like cells?
1. mammalian cell line
2. spherical shape
3. grow in suspension, not surface attachment
What are the advantages of cell cultures?
1. observe cell behavior without animal variations
2. maintain cells for generation= reproducibility
3. controlled growth environment
4. exposure to specific isolated reagents
What are the disadvantages to cell culture growth?
1. require standardized technique
2. time consuming
3. limited material
4. loss of original cellular mechanisms
Where can cell cultures be applied to life and sciences?
1. cell/gene function research
2. biological product formation
4. regenerative medicine
Why is protein purification important?
1. study structure and function of specific protein
What is recombinant DNA technology used for?
overexpress a specific protein making it easier to purify
What are the steps of sub-cellular fractionation?
1. mechanically blend tissue, to form homogenous mix
3. cell lysis
What techniques can be used to lyse cells?
1. osmotic shock
2. ultrasonic vibration
3. mechanical blending
What is ultracentrifugation used for?
1. separate organelles
What are lipid rafts?
1. PM domains high in cholesterol, sphingolipids, gangliosides
2. detergent insoluble
3. center for signal transduction
4. abnormal protein processing in neurodegenerative disorders
What are the different matrices used for column chromatography?
What is ion-exchange chromatography?
separation of compounds based on their attraction to the column based on ion charge.
What is gel-filtration chromatography?
separation of compounds based on size. Column has certain pore size, that compounds can get trapped in and retained. Larger particles drain faster, smaller drain more slowly
What is affinity chromatography?
similar to ion-exchange except charge is not used. Use the affinity of a substrate to an exnzyme. Stronger affinity equals longer retention time
How are genetically engineered proteins formed?
1. use of recombinant DNA technology
2.use tags to purify the protein and recognize specific epitopes
What is SDS used to do, when analyzing proteins in an SDS-PAGE experiment?
1. unfolds the proteins
2. attaches uniform negative charge
3. contains beta-mercaptoethanol to reduce disulfide bonds
What feature of proteins does SDS-PAGE use to evaluate different proteins in a solution?
1. the size of the denatured protein
What feature is critical to the analysis of proteins when using a 2-dimensional gel?
1. isoelectric points and the pH of the solution
If the pH is higher than the isoelectric point of a compound, which direction will it move?
the protein will migrate towards the + pole
If the pH is lower than the isoelectric point, which direction will the compound migrate?
the protein will migrate towards the - pole
If the pH is equal to the isoelectric point, which direction will the compound move?
it will not move.
What is Sirt3 and what happens if it malfunctions?
1. mitochondrial protein deacetylase
2. malfunciton= increased acetylated Sirt3 substrates
What is Western Blotting?
1. type of protein analysis that uses Ab-Ag interaction in order to determine a protein.
What is indirect immuno-cytochemistry?
1. type of western blotting that utilizes secondary Ab that are marked and will bind with a primary Ab-Ag interaction.
2. very sensitive method
How is ELISA useful in protein analysis?
1. provides protein detection and quantification
How is ELISA used to determine presence of Ag?
1. Ab-enzyme complex binds with the Ag, forming a color and absorbance level is then analyzed with machine
When would Indirect ELISA be used?
1. used to detect the presence of an Ab
2. used for HIV infection
3. use enzyme linked Ab react with Ag
What does an enzyme reaction suggest for an indirect ELISA test?
1. enzyme-linked antibodies were bound to human antibodies and implies the Pt has antibodies to viral antigen