Techniques Table

FRET application, how it works, results

-acts a as molecular ruler to measure distance between donors and acceptors -use fluorophores as donors and acceptors. UV (purple light) shines on sample. If see only purple, there is nothing there. If see green, donor is present, no acceptor. If see red, donor and acceptor are together. -flrorophores emit either red or green light, or will see purple (UV light) reflected back

Intercalating Agent application, how it works, results

-allow us to view nucelobases through stain or fluorescence in varying techniques -DAPI -Ethidium Bromide -see stain or fluorescence

Nucleic Acid Probes application, how it works, results

-allows us to look for specific genes on a chromosome -complementary sequence on probes (containing fluorophore) binds/anneals to the chromosome's complementary sequence (FISH STAINING) -fluorophore emits light at a specific gene sequence

Isoelectric Focusing application, how it works, results

-allows us to separate items based on isoelectric point (where protein is neutral and often precipitates) -gradient of pH is created, sample injected and moved down column, if pKa of the protein is lower than the pH solution it will be deprotonated. If pKa of the protein is higher than the pH of the solution is will be protonated. When changing pH, proteins will reach pI and precipitate in bands -bands of varying pI (isoelectric points) observed as proteins stop moving down the column

2D Gel Electrophoresis application, how it works, results

-allows us to separate protein based on pI and mass/molecular weight -run isoelectric focusing first, then an SDS PAGE on a gel by soaking in SDS. Gel will read in order of decreasing pI and decreasing mass -stainable bands of varying size and pI observed

ELISA application, how it works, results

-detection and QUANTIFICATION of a specific protein -paper from western blot is treated with a primary Ab (2 variable sites from: mouse, rabbit, rat) to bind to specific protein of choice. Then treated with secondary Ab (1 variable site, 1 site specific to primary Ab, enzyme attached to base, from horse, donkey, etc.) which has an enzyme that will react with colorless substrate -amount of color observed (from change in colorless substrate) is proportional to the amount of protein present

Sanger Sequencing application, how it works, results

-determines order of nucleotides in the DNA sequence -stops polymerization at the 3'-OH by tagging terminal nucleobase with a di-deoxy group, causing DNA to fluoresce at the base where polymerization needs to end! can then sort based on sequence length -observe fluorescence at the base where polymerization ends

Fluorescent Microscopy application, how it works, results

-for conjugated rings or planar structures -structure absorbs light, causing excitation and emission of a lower energy/longer wavelength photon shining back at the detector -fluorophores emit light back at detector and an image is produced

Liquid Chromatography application, how it works, results

-ion exchange, size exclusion, affinity chromatography -contains a mobile (liquid) phase and a stationary (plate, solid, column) phase. Ion exchange uses either salt or change in pH to elute; size exclusion uses slower wash speeds (wiffleball); affinity based uses special/specific elutant that acts as a binding ligand - purified/isolated liquid sample

3 types of Liquid Chromatography

-ion exchange:separate based on charges -size exclusion:separate based on size -affinity chromatography:separate based on binding capabilites

LC-MS-MS application, how it works, results

-liquid chromatography and mass spectrometry used to identify fragments of an unknown protein -contains a quadrupole, then can backstitch protein fragments LC, QUAD 1, QUAD 2, QUAD 3 -peptide fragments identified, can now backstitch

Cryo-EM application, how it works, results

-look at macromolecular complexes (typically structured/undynamic regions -cryogenically freeze sample on grid and shoot beam of electrons at sample (21 nm wavelength, 2nm resolution) -high resolution shadow images of complex

Light Microscopy application, how it works, results

-look at relatively larger samples -shine light onto sample from above or below -image of what sample looks like as-is (in normal light)

Hyperchromic Shift application, how it works, results

-measures structural integrity of DNA -when double stranded, DNA loses absorbance due to the large amount of H bonds. Denature strand of DNA using heat to break H bonds and increase the absorbance viewed -observe an absorption vs heat over time curve

Nucleic Acid Electrophoresis application, how it works, results

-molecular sieve, separation of sample by mass/size, cannot be used for proteins that don't have stable m/z -utilizes electrical current to pull negatively charged phosphate groups (in nucleic acids) up through the gel to the end (positive charge at end, negative at start - repels) -will see various bands separated by size, can stain to see more clearly

Western Blot (Ab Detection) application, how it works, results

-run after running a 2D gel to identify the proteins that were separated -paper with a positive charge on the other side is used to "stick" negatively charged/low pH proteins to paper -identify protein from the bands taken from a 2D gel electrophoresis

Differential Sedimentation application, how it works, results

-separates by mass and density -centrifugation to observe bands sorted by density -bands observed in order from least to most dense/massive (top to bottom)

SDS-PAGE application, how it works, results

-separates by size, can be used for proteins that do not have a stable m/z (mass to charge ratio) -boil to unfold protein, SDS wraps one time per 2-3 amino acids if denatured completely (creating false, stable m/z) -is gel electrophoresis, so will observe stainable bands that indicated mass/size or quantity

NMR application, how it works, results

-small proteins only, shows movement, shows protein in interaction in aqueous environments (IN SOLUTION) -magnet pulses waves at sample, protons tip off center and slowly spin back into place, amount of time taken for proton to spin back into place allows us to interpret varying side chain interactions of structure -can view in overlay over time by developing a stick-like model, blurred regions tend to be dynamic/mobile whereas structurally sound/immobile regions will be firm lines

X-Ray Crystallography application, how it works, results

-solve protein structure using crystal structures (typically structured/undynamic regions) -protein crystallized beams shot through sample and projected on a screen -shows dots where deflection points of the structure were, allows us to back calculated structure

Five techniques for protein separation

1. Differential sedimentation 2. Liquid chromatography 3. Isoelectric focusing 4. SDS-PAGE 5. 2D gel electrophoresis

Four techniques for protein structure elucidation (clarification)

1. FRET 2. Cryo-EM 3. X-Ray Crystallography 4. NMR

Two techniques for protein identifcation

1. LC-MS-MS 2. Western Blot (Ab Detection)

Two techniques for genetic sequencing

1. Nucleic Acid Probes 2. Sanger Sequencing

Three techniques for viewing samples/results

1. light microscopy 2. fluorescent microscopy 3. intercalating agemt

Technique for protein quantification

ELISA

Technique used for DNA structure analysis

Hyperchromic shift

LC-MS-MS Steps

LC:liquid chromatography to separate QUAD 1: mass spec, enrich for 1 m/z QUAD 2: collision cell, slams protein into N2 to break up QUAD 3: mass spec, identify peptide fragments

Technique for nucleic acid separation

Nucleic acid electrophoresis

LC-MS-MS Strengths and Limitations

S- allows identification of specific protein fragments L- expensive

Isoelectric Focusing Strengths and Limitations

S- allows us to gain information on a protein's isoelectric point; if successful, can now run a 2D gel electrophoresis L-lengthy analysis time

Cryo-Em Strengths and Limitations

S-2 orders of magnitude smaller than standard light microscopy (high res for small things!). Many images of sample in various orientations, pick out images, box, and cluster them L-destroys sample in the process, can't always shorten the wavelength without destroying sample (very high energy!), also not good for dynamic/mobile proteins as it freezes only one orientation

Sanger Sequencing Strengths and Limitations

S-accurate and reproducible L-can only sequence one fragment at a time; expensive and time consuming

Light Microscopy Strengths and Limitations

S-affordable, easy usuage L- poor resolution at high magnification

ELISA Strengths and Limitations

S-allows QUANTIFICATION of protein present, high specificity L-expensive

Nucleic Acid Electrophoresis Strengths and Limitations

S-allows us to accurately observe/compare sample fragment sizes to the predicted L-needs stable m/z ratio compares to length (1 nucleic acid = 1z); doesn't work for proteins (do not have a stable m/z)

2D Gel Electrophoresis Strengths and Limitations

S-allows us to gain information on the protein's size relative to its isoelectric point L-time consuming, bands ay contain multiple proteins (not just the one we want)

FRET Strengths and Limitations

S-allows us to measure interaction distances between individual molecules, allows us to see frequency and duration of interactions. Measures individual data points, doesn't average them L-low quantum efficiency

Hyperchromic Shift Strengths and Limitations

S-allows us to measure the structural integrity of DNA

SDS-PAGE Strengths and Limitations

S-can be used for proteins that do not have a stable m/z by creating a false one with SDS; if successful, can now run a 2D gel electrophoresis L-denatures protein sample

Western Blot (Ab Detection) Strengths and Limitations

S-can identify specific proteins within a 2D gel (allows us to differentiate between different proteins that may be within a single band) L-time consuming, difficult to reproduce results/procedure

Nucleic Acids Probes Strengths and Limitations

S-high sequence/binding specificity L-requires undegraded DNA; expensive

Fluorescent Microscopy Strengths and Limitations

S-high specificity (due to fluorophore probes) L- dependent on probes to fluoresce, anything untagged will be dark in final image

Intercalating Agent Strengths and Limitations

S-inexpensice, allows us to visualize results L-cancerous

NMR Strengths and Limitations

S-protein observed in close to native state (in solution), allows us to study structure and the dynamics of the protein, we can see movement L-cannot view large proteins, needs to be done on certain isotopes (need odd number of protons)

Liquid Chromatography Strengths and Limitations

S-purity increases L-yield decreases, need a liquid/soluble sample

X-Ray Crystallography Strengths and Limitations

S-very high resolution (0.2nm), close to seeing angstroms! Preferred for protein structures, solved 90% all known protein structures L-time consuming, not necessarily physiologically relevant (crystalized on one orientation only)

Differential Sedimentation Strengths and Limitations

S-was use to prove that DNA replicates in a semiconservative fashion L-must be in solution and able to centrifuge without damaging sample

How Ethidium Bromide Works:

Stains gels, cancerous, creates space between nucleobases, emits light in bands when pi. stacked with nucelobases, causes frameshift mutations through insertion

How DAPI Works:

fluroscent microscopy, creates less space between nucleobases, (not as cancerous)