Chapter 8: Methods in Cell Biology Flashcards
what are cell lines?
immortalized lines of donor cells crossed with cancer cells
why are immortalized cell lines crossed with cancer cells?
encourages constant cell proliferation and prevents them from dying out like normal cells
What is the BHK21 cell line?
a Syrian hamster fibroblast cell line
What are the downsides of immortalized cell lines?
can only replicate a few times to avoid phenotypic drifting
what are HELA cells?
highly robust human epithelial cancer cells that can easily contaminate other cultures
how are hybridomas created? (3)
- double nucleated heterokaryons created
- cells are allowed to proliferate
- hybrids are formed from nucleolytic crossing over
what are heterokaryons?
double nucleated cells
why are hybridomas used?
method for producing many pure cloned (myoclonal) antibodies; rather than polyclonal mixtures
what speed would you spin to get whole cells, nuclei, and cytoskeleton?
low speed
what speed would you spin to get mitochondria, lysosomes, and peroxisomes
mid speed
what speed would you spin to get microsomes and vesicles?
high speed
what speed would you spin to get ribosomes, viruses, and other large macromolecules?
very high speed
this cell biology method passes sample through media by constantly applying solvent
column chromatography
why is column chromatography used?
separates sample into fractions based on ions, affinity, size, etc.
what are the steps for using epitope tags?
- epitope tag is introduced to target gene
- gene is introduced to cell
- produces epitope-tagged protein
- protein can then be used downstream
what are two ways epitope-tagged proteins are used?
- rapid purification from cell
- visualized using fluorescence
what is the problem with epitope-tagged proteins
tags can prevent proper folding; they can only be used for certain proteins (observation changes function)
what is the purpose of SDS page electrophoresis?
determines molecular weight of whole proteins/subunits
why is the substance SDS used in SDS page electrophoresis?
breaks disulfide bridges (encourages subunit formation) & effects charge
this is a method of gel electrophoresis that separates samples based on isoelectric pH point
isoelectric focusing
this is a combination of both SDS page and isoelectric focusing; creates two axes of visualization
2D electrophoresis
what are some alterations that must be made to 2D electrophoresis to aid in visualization
- need large amounts of protein
- use silver-based dyes
what are restriction enzymes typically used for?
generally used to customize bacterial plasmids
what is the scientific purpose of using restriction endonuclease?
capable of recognizing and chopping up non-self DNA so that new sequences can be inserted