Lab F

Introduction:

  • Two gels will be used

    • Will wants to figure out the size and number of individuals subunits of a given protein using the SDS-PAGE technique

    • We will also determine the quality and the quantity of the PCR product we made use gel electrophoresis and the spectrophotometry

Part A:

  • Proteins have different structures

  • Gel Filtration: provides an estimate of the molecular wieght of native state proteins

  • SDS with a reducing agent separates protein subunit and separate through a polyacrylamide gel electrophoresis

How does the SDS-Page technique work?

  • referred to as denaturing electrophoresis

  • molecular weight is estimated comparing the migration of a denatured protein/unfolded protein/

  • SDS → anionic detergent sodium dodecyl sulfate

    • Is mixed with a reducing agent to create a loading buffer meant to break down disulfide bonds

    • SDS disrupts noncovalent interactions between subunits of proteins which causes protein unfolding

    • proteins will become negatively charged with SDS

  • proteins are added to the gel the same as DNA and will run from negative to positive based on their size but will use a polacrylamide gel instead

  • Acqua Stain will be used to stain the proteins in order to visual their runs

Part A Procedure:

  • get a protein samples from the TA

  • denature them in the heat block and then load it into a well

  • run gel at 250 V for 25 minutes and then stain with acquastain

  • will paste the graph on the worksheet

  • will use the curve to determine the size of an unknown polypeptide

    (confused)

Part B: Agarose Gel Electrophoresis and Spectrophotometry

  • Electrophoresis: provides a qualitative overview of DNA purity

    • allow us to know whether amplified DNA is the correct size

    • agarose gel will be used here instead

  • Gels will be stained with gel green which will intercalate between base pairs and will be visualized under a blue LED lights

  • Spectrophotometry: provide a quantitative measure of the DNA

    • absoroption of 260/280 indicated good nucleic acid purity

    • to estimate the concentration of DNA, multiply the concentration of the sample by 50

  • we will be determine the amount of DNA in the PCR product and will use the ratio to determine the nucleic acid purity

Part B Procedure:

  • get a tube and mix the pertaining liquids and flick it, load into the gel

    • lowkey was confused there watch the video for help

  • Spectrophotometry to check DNA concentration

    • make a cuvet and then mixe with a pipette and read it with the spectrophotometer taking the 260 and 280 readings

warnings:

  • some irritations from gels and buffeds

  • gel green can also cause irritation

  • TBE buffer as well

  • everything goes in the regular trash

Video 1:

questions:

When is Text 2 for the Human Physiology lab due

  • Friday of Week 6 at NOON

The protein materials in LS7L (Instruments & Materials) are identified by the color 'orange'.

  • False

    • DNA = Orange

    • Proteins = Green

  • When loading a gel, why is it important to only push to the first stop of the pushbutton on the pipetter?

    • Pushing to the second stop may cause bubbles to blow your sample out of the gel well.

According to the lab safety sheet, which of the following is a potential hazard you will face in the Agarose and Polyacrylamide Gels lab?

  • Chemicals causing respiratory tract, eye and skin irritation

  • You will be loading two different types of gels. Which gel setup is shown here and what are we using it for?

    • PAGE / Protein (standing up thing that is labeled green)

      • Remember the Polyacrylamide gel will be used with the protein

  • This is the other type of gel, the agarose gel. What do we use it for in lab this week?

    • PCR product size

      • Agarose gel will be used with the Gel electrophoresis one

  • DNA will move through the gel towards the positive electrode because...

    • DNA is naturally negatively charged and the negative charge is attracted to the positive electrode.

      • we will not be treating the DNA with SDS, that is inly for the protein

  • looking at both quantitative and qualitative success of PCR

  • you will start by loading the protein sample where the TA will start running the DNA

  • you will get the DNA from the 96 well from last week and then loading it in the DNA gel

  • will analyze a mark protein

Part 2 of the Lecture Videos:

questions:

  • You add 5ul of your PCR product solution into 495 ul water. What is the dilution factor?

    • 100

      • we are supposed to dilute our DNA once we collect the sample from the well

  • If the ratio of OD260/OD280 is 1.5 the DNA prep is pure

    • False should be around 1.8

  • The concentration of the DNA can be determined by using the OD280 reading.

    • False, it should be the 260 reading

  • You read an Absorbance of 1.00 at 260nm & 0.56 at 280nm. The DNA sample is…..

    • Pure

  • You read an Absorbance of 1.00 at 260nm & 0.56 at 280nm. What is the DNA concentration in the cuvette?

    • 50ug/ml (microgram/mililiters) cause this will be equal to 0.05 milligram/militers

  • The DNA concentration in the cuvette is 50ug/ml. When you prepared your DNA sample for the spec reading you used 5ul from your PCR reaction and added 495ul water. What is the DNA concentration in the PCR reaction?

    • 5mg/ml

  • What is the size (bp) of the Extended Hypervariable Segment I (HVSI)?

    • 651 BP

  • What size (bp) do we expect the PCR product to be?

    • 549 = 16520 - 15972 + 1

  • How to determine the purity of DNA prep

  • Determine concetration of DNA Prep

  • how to determine molecule length such as PCR product length

  • once we dilute we will take the reading on the spectrophotometry

    • reading done at 260 and 280

      • ratio about 1.8 good job isolating the DNA

      • less than 1.8 then protein is involved

  • The ratio between 260/280 of the DNA will determine the purity of the DNA prep

    • you will get the ratio once you have diluted the DNA with 495 of water and you take the reading

  • DNA concentration in the cuvette is determined by multiplying the 260 reading by 0.05

  • dna should equal 500 micrograms or 5 miligrams since the dilation factors was 100 so i got this from multiplying 50 and 0.05 by 100

  • molecule length: 651 base pairs

    • highest # - lowest # + 1 ( will give you the number of bp in a region

  • a success PCR will have the product at around 549 in all lanes with faint primer dimers

  • no bands and only once successful PCR means a failed PCR

    • might have failed due to having no DNA present?

part 3.1 (proteins)

questions

  • A protein dimer is an example of the

    quarternary-structure of a protein. An Amino-Acid-Sequence is an example of the primary-structure. An alpha-helix is an example of a secondary-structure and a protein-domain is an example of the tertiary-structure.

  • Which if these molecular interactions is a covalent bond?

    • Disulfide bond

  • SDS (Sodium-dodecyl-sulfate) breakes cystein-linkages

    • False, the BME does this

  • Which type of protein gel will run proteins while keeping their enzymatic function intact?

    • Native Gel - Just buffer

  • In Size Exclusion chromatography larger molecules come out later than smaller molecules

    • False

  • Protein size is measured in

    • kilodaltons, sicne theyre so large

  • We have different molecular weight standards

  • homo-tetramer

    • will have the same subunits (4)

  • homodimer

    • will have two of the same subunits

  • Monomer

    • will have a single subunit

protein structure:

  • primary: single piece of protein

  • secondary: local interactions between amino acids

  • Tertiary: interactions between domains

  • quaternary: interactions between subunits

different types of bonds in protein structure

  • ionic: salt bridges?

  • hydrogen bonding between side chaings

  • dissulfide, covalent, and usually dont unfold

  • SDS and heat will be used to unfold a protein without cysteine bridges and will make a single unit

  • BME will work the same way but for proteins that have cysteine bridges

  • estimating a protein mass

    • there are different types of gel used to do this

      • native gel: keeps the protein intact

      • SDS: unfolds protein without cysteine bridges

      • Reducing Gel: proteins with cysteine bridges will unfold

  • proteins measured in kilodaltons

examples of protein purification

  • ultracentrifugation

    • separation based on size, shape, density

  • column chromatography

    • protein is added to a solvent with a lot of buffer which will dilute and separate?

    • separation by size

      • large come out faster

      • smaller molecules get trapped and come out slower

  • Choose the correct statement

    • My unknown protein can have one or more subunits

  • you can monitor each step of purification through the gel electrophoresis

lecture video 3.2

   questions

  • In lab, protein will move through the gel towards the positive electrode because...

    • The protein is treated with SDS, which creates a uniformly negative charge in the molecule that is attracted to the positive electrode.

  • Choose the correct statement about your lab activity in the gels lab

    • You will be given a protein of known molecular weight (predetermined by gel filtration on the native molecule) but unknown subunit composition.

  • For this lab, we will use what type of stain for the PAGE gel?

    • acquastain

  • The loading dye's function is

    • to allow you to visualize how far the samples have run on the gel

  • Which of these functions shown in the image has the best fit for the data?

    • Exponential, since its the curve that fits most of the data points

  • An SDS-PAGE reducing gel

    • has SDS & beta-mercapto-ethanol and will break down disulfide bonds

  • You run a reducing SDS-PAGE of a protein with a known MW of 260kD. You get three bands (sizes 30kD, 40kD & 50kD).  What is the protein composition?

    • 3 of 50kD - 2 of 40kD - 1 of 30kD

      • will need to add up to 260 K

  • You run a non-reducing SDS-PAGE of the same protein. You get two bands. Why do you get only two bands?

    • Cysteine bridges between two 40kD and one 30kD Subunit

  • protein size estimation

  • PAGE gel setup

    • aqua stain will stain the protein in the gel after 15 minutes when added

  • you will measure the bands and create a curve out of them

  • we will have standards to compare are unknown to

    • We will first measure the know, and then the unknown, make the curve to get the protein estimate

  • Molecular weight has been determined by running the protein through the native gel

  • reducing gel will have BME

lecture video 3.3

questions

  • You need to separate very large proteins (>200kD) using a native Polyacrylamide Gel Electrophoresis (PAGE). Which percentage of polyacrylamide would you choose for optimal separation?

    • 5 %

  • Why is it necessary to run standards alongside your samples every time in a PAGE gel?

    • To account for variations in gel and running conditions.

  • Choose the correct one

    • In LS 7L we use GelGreen to visualize the DNA, because it is a safe alternative to ethidium-bromide which is toxic

  • will have gel parameters

    • voltage

    • change the percentage of gel

    • how long the gel is ran for

  • gel will run depending on condition of parameters

  • with a higher percentage of the gel, the larger molecules will get stuck and not travel far

  • the higher the voltage the faster the protein will run

    • it could cook the gel

    • better to run with a lower voltage

  • time, running too early is bad when making the curve

    • running for too long then the bands will fall off the gel

  • gel is stained after its ran in the PAGE

  • in the agarose gel then stain is already there

  • we will used acquastain with the protein

  • we will use gel green with the DNA

  • the horizontal setup

    • Agarose / DNA / PCR Product Size / GelGreen

  • the vertical set up

    • PAGE / Protein / Protein Composition / AcquaStain