BIOL 325 RESOLUTION/DETECTION OF NA AND PROTEINS STUDY GUIDE

why should you use A320 for DNA?

it tells/subtracts out any issues with purity and adjusts for turbidity

why is 230 nm better for DNA?

other things like RNA and aromatic amino acids are absorbed at 260 nm

how do you find the concentration for DNA with spec?

A260 - A320 x DF x 50 micrograms/mL

what is the number(s) for good quality DNA?

1.7-1.9 at 260/280

how do you evaluate DNA purity?

A230/A320

what are the dyes that are used for fluorometry for DNA?

• DABA

• Hoechst 33258

• PicoGreen dsDNA

• oligogreen ssDNA

what are the dyes that are used for fluorometry for RNA and what should you do first?

• ribogreen

• sybergreen 2

• REMOVE DNA FIRST

how can nucleic acids be detected in whole cells? how about in isolated nucleic acids? (written response question)

• in whole cells, you can stain the DNA using DAPI and Hoechst which are blue dyes.

  • DAPI is for fixed cells and stains the nuclei of cells where the DNA resides and can tell which stage the cells are in

• for isolated nucleic acids, you can use gel electrophoresis to visualize the isolated NA and find the approximate concentration, yield, and purity

  • use spectrophotometry to find the concentration, yield, and purity

what is propidium iodine?

a stain that differentiates living and dead cells and is not membrane permeable

what is electrophoresis?

movement of molecules by an electric current

how do you make percent solutions?

g (whatever is needed) x 100 mL

name various properties of proteins and nucleic acids that can be exploited for electrophoresis separation (written response question)

• proteins = molecular weight for denatured proteins

  • 3D structure for whole proteins

  • charge (smaller proteins = negative and longer proteins = negative)

• nucleic acid = size/length of nucleotide sequence

in gel electrophoresis, how do the fragments migrate?

• smaller fragments run through the gel faster, migrating a great distance

• larger fragments run through the gel slower, migrating a slower distance and is closer to the well (gDNA)

name the two different polymers used in gels and their percentages (written response question)

• the two polymers used in gel are:

  • agarose for DNA and polyacrylamide for DNA or proteins

• 0.5-5% agarose

• 3.5-20% polyacrylamide

what is the role of a comb in gel electrophoresis and what are the two different types of combs?

role of comb = create wells in the cast gel for sample loading and the depth/diameter of the comb gives the total loading volume in wells; use the proper comb (wells) and gel size

• regular comb = wells are separated by “ear” of gel

• houndstooth comb = wells immediately adjacent

how does polyacrylamide work for a sample to run through its gel?

two forms of acrylamide are present with a catalyst added which makes a complex matrix that acts like a sieve for the sample to work through

what is the role of running buffers and name the buffers and their purpose (written response question)

role of running buffers = carries the current and protects the sample during electrophoresis; current moving through the gel separates the molecules

• tris borate EDTA (TBE) = resists pH change well

• tris acetate EDTA (TAE) = faster but expires

• tris phosphate EDTA (TPE) = gets hot

what electrophoresis buffer is used for RNA?

10 mM sodium phosphate or MOPS buffer

what are the denaturing agents used for proteins?

• formamide (common)

• urea (uncommon)

• methylmercurichydroxide (toxic)

what do recirculate buffers prevent?

ionization and pH drift

what is the role of ladders/markers for DNA and proteins? (written response question)

role of ladders = to distinguish between two different DNA/RNA/protein fragment sizes and approximately see the molecular weight

what is the role of stains and name the two stains in class and their purpose (written response question)

role of stains = to visualize the sample and can be added before loading or after the gel has gone through electrophoresis; can be used quantitatively

• ethidium bromide = intercalating agent meaning it will insert itself into the double-stranded DNA but is carcinogenic

  • use fluorometer to see the sample

• SyBr green or SyBr gold = used for single or double stranded DNA or RNA and has lower health concerns

  • use fluorometer to see the sample

when you are running a gel what would you load a DNA sample with?

loading/tracking dye to visualize the sample and give the sample density to fall into the well

when you are running a gel for RNA, what would you load it with?

loading dye with DMSO to break the disulfide bonds in RNA’s tertiary structure

when running a gel for proteins, what would you load it with?

loading dye with SDS to denature the proteins

how is a band formed in gel electrophoresis?

the collection of molecules that have moved through the gel at their speed of migration

complete this sentence: the higher the percentage of agarose and how does this relate to what is isolated?

• the slower the molecules will move and will be closer to the well

• choose the percentage of gel depending on what needs to be isolated and the smaller the fragment the more agarose needed

what is the difference between in native and denaturing gels? (written response question)

• native gels are ran for the protein’s 3D shape

  • molecules migrate based on size, shape, and charge

• denaturing gels isolate to the primary structure of proteins (chain of amino acids)

  • molecules migrate based on the proteins molecular weight

what are the three research techniques for protein gels?

• identifying mutant proteins

• evaluating toxins from microbes

• finding spike protein variants in virus

what is polyacrylamide gel electrophoresis (PAGE)?

• acrylamide + a crosslinker, methyl bis-acrylamide

• is a synthetic polymer meaning that is made in labs to beautifully separate proteins

why should you buy commercial gels instead of making your own?

avoid neurotoxins and to improve the quality

what is PAGE’s polymerization catalyst?

ammonium persulfate (APS) + (TEMED)

what does polyacrylamide gel electrophoresis resolve in a DNA sample and proteins?

• 1 base pair difference in a 1 kilobase DNA fragment (0.1% difference) meaning that PAGE can separate very small differences in DNA

• PAGE can resolve proteins whose molecular weight are from 0.5 to 50kDa in a single lane meaning that the proteins can separate clearly and identify the proteins in that size range

what is the history of acrylamide?

• formed through something rich in sugars and something rich in proteins that have asparagine in plant-based foods including potato and cereal grains

• also formed through high-temp cooking like frying, roasting, baking and anything burnt like toast or burnt marshmallows

• causes cancer in animals in high doses so in 2010 FDA classes acrylamide as a health concern

what are the uses of acrylamide?

• sequencing (old school)

• mutation analysis

  • can use for DNA to see 1 nucleotide difference

• nuclease protection assay

• blotting

• acrylamide is thin (30 micrometers like notebook paper) so can change sieving properties to get amazing resolution

how do you prepare protein for electrophoresis?

1. isolate protein from sample of interest via homogenization

   • density-gradient centrifugation is used to collect specific fractions

2. add lysis buffer

   • often SDS is used to denature proteins and add negative charge proportional to mass (bigger mass = bigger negative charge)

3. boil w reducing agent (beta ME or DTT) to break disulfide bonds to reduce it to its primary structure (denaturing conditions only)

4. add BTB tracking dye/loading buffer to see sample and visualize it

when spinning the centrifuge at a low speed for density-gradient centrifugation, what is in the pellet?

nuclei and cytoskeleton

when spinning the centrifuge at a medium speed for density-gradient centrifugation, what is in the pellet?

mitochondria, lysosomes, peroxisomes

when spinning the centrifuge at a high speed for density-gradient centrifugation, what is in the pellet?

microsomes, small vesicles

when spinning the centrifuge at the fastest speed for density-gradient centrifugation, what is in the pellet?

ribosomes and large macromolecules

what is the common gel for protein electrophoresis?

SDS PAGE gel

what do proteins travel based on when SDS denatures them?

molecular weight NOT by their 3D structure

what does polyacrylamide do for proteins?

separates large proteins from small ones in a small area

what is the gel concept for protein electrophoresis?

• has two layers of gels and needs great resolution so every millimeter of the gel actually separates the proteins from each other

• the stacking gel is high in polyacrylamide and lower pH than the resolving gel so the glycine has a positive charge

• both the gel and buffer contains glycine (aa) with no charge at pH 7

  • glycine is in the buffer to push the sample down because proteins are often in vertical equipment

• in resolving or separating gel at pH 8.8 so glycine becomes negatively charged

what is the procedure for PAGE for protein resolution?

• before sample is loaded, add anode and cathode buffer

• sample is denatured by SDS and loaded into the gel

• add power source for current to separate protein bands

why perform gel electrophoresis?

if trying to analyze one particular DNA or protein in a giant mixture of DNA or protein, you would need to separate them based off their size/length of the nucleotide sequence

what would happen if distilled H2O was used and not buffer?

nothing to carry the current so the DNA/RNA/proteins would not flow down

how can you separate nucleic acids?

use agarose gel and the length of the nucleotide sequence is separated

how can you separate proteins?

use polyacrylamide gels; separate with density gradient centrifugation (MW or charge)

what are the advantages for coomassie blue dye?

• low cost

• simple procedure

• easily modified for fast or highly sensitive staining

• more suitable for quantitative analysis than silver staining

what is silver stain more sensitive to?

ssDNA, dsDNA, RNA, AND proteins but its toxic in its raw state and VERY sensitive

for gel electrophoresis, what are the two formats?

horizontal/submarine or vertical

how does the angle of the electrode affect the current in gel electrophoresis?

the angle of electrode is what forces the currents conductivity in one direction

what method would you use to resolve very large DNA fragments? be DETAILED (written response question)

the method that I would use to resolve very large DNA fragments would be CHEF.

• CHEF resolves up to 9 Mb and resolves DNA over a wide range of molecular weight in a straight line.

• CHEF uses controlled electric fields that reorient the direction of the large DNA molecules by turning certain electrons on and off at a predetermined angle for better separation.

what is pulsed field gel electrophoresis (PFGE)?

• can change the direction of the DNA depending on its size and shape

• DNA fragments move lengthwise and crosswise in agarose

• can resolve up to 6 mega base pairs

what can pulsed field gel electrophoresis be used in other than resolving very large DNA fragments?

• is a powerful tool in control, prevention, and monitoring diseases so this is necessary for vaccine preparation

• can be used to see similarities and differences in many different samples so can be used as a molecular weight marker

• in computers there is a large data-base of PFGE gels

  • individuals can tell which gene patterns are the most virulent when comparing samples

  • can also create phylogenetic trees (sees if they have a common ancestor)

what is field inversion gel electrophoresis, a type of PFGE?

• uses alternative positive and negative poles

  • pushes negative to postive and positive to negative for unequal amounts of times to allow itself away from its neighbors in similar size

• resolves up to 2 mega bases (2 million bases)

• pulse controller is added to standard apparatus

what are the advantages of field inversion gel electrophoresis (FIGE)?

• no fancy equipment necessary

• simple to perform

what is transverse alternate field electrophoresis (TAFE), another type of PFGE?

• has fixed polarity = electrodes are moved but the currents are being alternated (↘↙)

  • the alternations are constant and equal

• vertical gel

• resolve up to 6 M bases or 2-6000 kb

• used to compare and contrast bands for strains and ancestry

• ex. genomic DNA of 6 thermophilic bacteria digested w Xbal from hot spring

  • digest with restriction enzyme to chop DNA into fragments then use pulse field to separate the fragments from each other

what is rotating gel electrophoresis (RGE), another type of PFGE?

• gel is rotated with fixed poles

• isolated 4Mb but takes 1-2 days

  • good b/c it slowly separates the DNA

• electric field is constant and unchanged and with the use of a single set of electrodes, direct separation of molecules is the result

• faster than the other methods

what is capillary electrophoresis (CE)?

• ions are separated based on their electrophoretic mobility using applied voltage and their mobility is dependent upon molecule charge, size, and viscosity

• the rate at which the particles move is directly proportional to the applied electric field which means the greater the field strength, the faster the mobility

• the molecules are moved through a thin capillary tube and used to separate nucleic acids, RNA/DNA, and proteins

• solvent is in sample

would a neutral species be affected in CE?

no because no charge

what is the purpose of the detector in CE?

a specific wavelength of light that evaluates the sample as it moves through the detector and produces outputs such as a nucleotide sequence

when CE is used to separate fragments what can those be used for?

• PCR

• sequencing

• cloning

how do you set up capillary electrophoresis?

• one end of the tube will be in the anode and the electric current is sent here too and electrons will be pushed in the direction toward the cathode

  • electrons pushed away from the anode —> anode becomes positively charged and electrons forced towards cathode —> cathode becomes negatively charged

• after CE has run, the big heterogenous mix of samples in it will have different sizes and charges, and the more negative a sample is, the closer to the anode it will be, and the more positive the sample is, the closer to the cathode it will be

  • the bigger the molecules in the sample = will impede their movement throughout it

• the solvent carrying the protein/nucleic acid and are separated by charge to mass ratio

describe the basics of capillary gel electrophoresis and what are its advantages over slab gels (written response question)

method where a gel-like matrix, like polyacrylamide which is used for sieving, is in a capillary tube and separates by solute size/charge ratio but also detects small sequence changes

• automated where a needle within a computer does the injection into the capillary gel tube and adds the sample slowly by charged platinum electrode where only a small sample amount is required

• CGE minimizes solute diffusion meaning that it has a tight, punctate spectrum, heat does not affect the sample by slowing molecules down and the gel prevents the solute from being absorbed into the capillary walls

its advantages over slab gels are that it is automated, more rapid, more precise, and only require a small amount of sample

what are the uses of capillary gel electrophoresis?

• suspect identification

• establishing lineage

  • sees lineage of a cell

• bone marrow analysis

• genome instability

• clonality testing

what is serum protein electrophoresis (SPEP)?

• used to separate various proteins in serum

• agarose gel is used with the same sample to visualize the sample

• separation results in distinct bands that represents different proteins like

  • albumin —> needs to have a lot of albumin

  • alpha globulin

  • beta globulin

  • gamma globulin

• can be used to see multiple myeloma because there is a huge peak of another globulin

what is apolipoprotein (ApoE) and what are the risk factors for the different ApoE’s?

• is a major component of lipoproteins, and participates in transport and clearance of lipids

• ApoE4 = risk factor for Alzheimer’s and other neurodegenerative diseases

• APoE2 + ApoE4 = increased risk for cardiovascular disease

what is western blot (immunofixation)?

• very sensitive technique to detect proteins using antibodies

• used when there is not enough protein to sustain it so antibody is used and some kind of colorimetric output

• loading controls are important with western blot as it ensures the amount of protein across all lanes is consistent

describe the basics of IEF, what properties does it separate proteins by? (written response question)

it is the primary technique for proteomics to work and the properties it separates by are PI value and molecular weight

• first, the sample is immobilized by pH gradient of polyacrylamide or agarose

• sample is placed on top of PAGE gel which is on a horizontal axis and separated by PI where protein is going to be uncharged

• when sample is already partially separated, it is separated vertically by molecular weight in kDA

can separate up to 10,000 proteins in a single gel

what might IEF be used for in a diagnostics lab? (written response question)

IEF is used in diagnostics laboratories to separate proteins based on their PI and their molecular weight. This allows the detection of genetic disorders like abnormal protein isoforms.

IEF can be used to detect autoantibody-mediated depletion of Still’s Disease