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EXPERIMENT
文章出處
  蔡豐州醫師
日期&閱覽
 張貼日期:2005/1/12  閱覽次數:4621

@ SDS-PAGE (sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis):
(1) Function:
Establishing protein size
Protein identification
Determining sample purity
Identifying disulfide bonds
Quantifying proteins
Blotting applications
(2) SDS is a anionic detergent--- to take the protein from its native shape, which is basically a big glob, and open it up into a linear piece, giving them linearity and equal negative charge(SDS is anionic). This will allow it to run more efficiently down the gel.

The SDS has a hydrophobic tail that interacts strongly with protein (polypeptide) chains. The number of SDS molecules that bind to a protein is proportional to the number of amino acids that make up the protein. Each SDS molecule contributes 2 negative charges, overwhelming any charge the protein may have. SDS also disrupts the forces that contribute to protein folding (tertiary structure), ensuring that the protein is not only uniformly negatively charged, but linear as well.

(3) The polyacrylamide gel is a cross-linked matrix that functions as a sort of sieve to help "catch" the molecules as they are transported by the electric current. Once an SDS-PAGE gel is run, you need to fix the proteins in the gel so they don't come out when you stain the gel. Acetic acid 25% in water is a good fixative, as it keeps the proteins denatured. The gel is typically stained with Coomasie blue dye R250, and the fixative and dye can be prepared in the same solution using methanol as a solvent. The gel is then destained and dried.
(4)
EDTA is a preservative that chelates divalent cations, which reduces the activity of proteolytic enzymes that require calcium and magnesium ions as cofactors.
The tris acts as a buffer, which is very important since the stacking process in discontinuous electrophoresis requires a specific pH.
Glycerol makes the sample more dense than the sample buffer, so the sample will remain in the bottom of a well rather than float out. The dye allows the investigator to track the progress of the electrophoresis.
SDS, DTT, and heat are responsible for the actual denaturation of the sample. SDS breaks up the two- and three-dimensional structure of the proteins by adding negative charge to the amino acids. Since like charges repel, the proteins are more-or-less straightened out,
immediately rendering them functionless. Some quaternary structure may remain due to disulfide bonding (covalent) and due to covalent and noncovalent linkages to other types molecules. By the way, another name for SDS is lauryl sulfate. Your shampoo may contain lauryl sulfate - now doesn't that inspire confidence in the product?

@ resolving gel
    stacking gel
@ cell wash with cold PBS 2 times
--- lysis buffer (M-PER) shake 5 mins and scrape into 1.5cc tube
<ps> RIPA need on ice
<ps> cell grown in 10cm plates typically contain 107 cells(50mg) and yield 3 mg total protein
--- votex 1 time/10 mins
--- centrifuge 12000g, 10 mins
--- take supernatant into new 1.5 cc tube

@DNA extraction
D
etergent & Salt, eNzymes (meat tenderizer), Alcohol

@ RNA extraction
1. TRIzol reagent (GIBCO BRL No.15596-026, 15596-018)
This reagent is pre-mixed solution of 50% phenol and 47% GTC. The protocol is very simple and good to avoid RNAse contamination. So I will recommend this if you are not the expert for RNA extraction. I usually get 40-60 micro g of total RNA from 4,0000000 cells (2,30 micro l of pellet)
** TRIzol® Reagent is very toxic and should only be used with
caution. Work only in a fume hood with proper personal protective
equipment (gloves, eye protection, etc.). TRIzol® waste is
hazardous and should be handled and disposed of according to
institutional hazardous waste protocols.
2. REX Total RNA Extraction Kit (USB No.75000)
Actually, all the solutions in this kit are preparable. So I'm following the protocol only. This protocol is not so simple as that of TRIzol, but will give you cleaner RNA. Yield; 1-1.5mg of total RNA from 1ml of Chlamy pellet by this method.

@ Cell Proliferation assay
(1) MTT Cell Proliferation Assay
The MTT Cell Proliferation Assay is a colorimetric assay system which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. After incubation of the cells with the MTT reagent for approximately 2 to 4 hours, a detergent solution is added to lyse the cells and solubilize the colored crystals. The samples are read using an ELISA plate reader at a wavelength of 570 nm. The amount of color produced is directly proportional to the number of viable cells.

(2) [3H] - Cell Proliferation assay
-- Measurement of [3H] thymidine incorporation as cells enter S phase has been a traditional method for detection of cell proliferation. Subsequent quantification of [3H] thymidine is performed by scintillation counting of autoradiography. This technology is slow, labor intensive and has several limitations including the handling and disposal of radioisotopes and the necessity of expensive equipment.
-- synchronize the cells in a low growth state, add the protein, incubate for 18 hrs followed by a 6 hrs thymidine pulse and then use 5% TCA and 0.5N NaOH/SDS to solubilize the cells and measure the CPM/DPM in a scintillation counter

(3) BrdU Cell Proliferation Assay
A well established alternative to [3H] thymidine uptake has been demonstrated by numerous researchers. In these methods bromodeoxyuridine (BrdU), a thymidine analog, replaces [3H] thymidine. BrdU is incorporated into newly synthesized DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, BrdU is detected immunochemically allowing the assessment of the population of cells which are synthesizing DNA.

@ Cytotoxicity Assay
Two possible cytocidal mechanisms have been proposed for cell-mediated cytotoxicity:
(i) the apoptotic mechanism, in which the effector cell triggers an autolytic cascade in the target cell and the genomic DNA fragments before cell lysis; and
(ii) the lytic mechanism, in which lytic molecules, notably perforin, are secreted by the effector cell into the intercellular space and polymerize to form pores in the target cell membrane, leading to cell lysis.
--- It quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a 30 to 60 minute coupled enzymatic assay that results in the conversion of a tetrazolium salt into a red formazan product. The amount of color formed is proportional to the number of lysed (dead) cells. Visible wavelength absorbance data is collected using a standard 96-well plate reader. Thus, the assay can be used to determine the total number of viable cells in a given sample.
          The modified MTT assay works similarly, but uses LDH to determine cell viability rather than cell death.  
 
@ SELEX(systematic evolution of ligands by exponential enrichment)
In 1990, the laboratories of G. F. Joyce (La Jolla), J.W. Szostak (Boston), and L. Gold (Boulder) independently developed a technique which allows the simultaneous screening of more than 10exp15 individual nucleic acid molecules for different functionalities. This method is commonly known as "in vitro selection", "in vitro evolution" or "SELEX" (systematic evolution of ligands by exponential enrichment). This novel technique is gaining more and more importance as an extremely useful tool in molecular biology. With the in vitro selection-technique large random pools of nucleic acids can be screened for a particular functionality, such as the binding to small organic molecules, large proteins or the alteration or de novo generation of ribozyme-catalysis. Functional molecules ("aptamers" a linguistic chimaera composed of the latin aptus = to fit and the greek suffix -mer) are selected from the mainly non-functional pool of RNA or DNA by column chromatography or other selection techniques that are suitable for the enrichment of any desired property (see scheme).
-- The most common method for the isolation of nucleic acids
with an affinity to a certain target molecule uses affinity
chromatography
with a solid matrix to
which the target molecule of interest is immobilized.




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