Membrane Transport and Lab Tests by Abigail Schofield https://padlet.com/aschofield242/u8vitovr5ejf181r en-us 2023-03-02 18:57:14 UTC 2023-03-03 13:45:45 UTC hello@padlet.com Cell Membrane aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501591011 - Maintains homeostasis by controlling what goes in and what goes out
- Phospholipid bilayer
     -Heads of lipids: Hydrophilic polar
     -Tails of lipids: Hydrophobic nonpolar

]]> 2023-03-02 18:59:26 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501591011 Passive Transport aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501591922
  • Molecules move from high to low concentration
    • Moves WITH the concentration gradient
  • Simple Diffusion
    • Very small nonpolar molecules can pass through the phospholipid with no help or energy
    • Molecules move from high to low concentration

    • Moves WITH the concentration gradient


    • Facilitated Diffusion
      • Uses a transport protein because molecule may be too big or polar
      • Doesn’t require energy
      • May require a stimulus (signal, usually an ion)


    ]]>     2023-03-02 19:00:02 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501591922     Active Transport aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501598042
  • Movement from low to high concentration
    • Moves Against the gradient
  • Requires ENERGY
    • ATP!
  • ATP energizes the protein channel to transport molecule against the concentration gradient

    • Endocytosis(3 different types)
      • A type of active transport where the substance fuses with the cell membrane and brings the substance inside the cell in a vesicle

        1. Phagocytosis
          • Used by amoebas to engulf a substance
        2. ReceptorEndocytosis
          • Special receptor proteins capture a specific target molecule
        3. Pinocytosis
          • Intake of liquid

    • Exocytosisis another form of active transport
      • Cell expels/releases/removes material OUT of the cell
      • This can include releasing neurotransmitters or proteins


    ]]>     2023-03-02 19:04:32 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501598042     Gel Electrophoresis aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501602138
  • Separates DNA by size
    • Shorter DNA travels further through gel
  • Utilizes Agarose
    • Agarose is very porous
  • Needs a buffer
    • This allows the electric current to travel through the gel
  • Uses:
    • Crime scenes
    • Paternal/Maternal testing
    • Comparing species
    • Identifying/observing certain diseases
  • When separating DNA, it all has the same charge
    • DNA will move from negative to positive ends!
  • DNA Ladder:
    • Known DNA sizes that are used as a standard to compare other samples 


    • ]]>     2023-03-02 19:07:28 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501602138     Gel Electrophoresis PT 2 aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501602867
  • How does it work?
  • Each line on the gel is a band of DNA. 
    • Thicker bands mean there was a lot of DNA in that specific band
  • DNA samples that have similar banding patterns will be more closely related
    • Closely related organisms have similar DNA, therefore their banding patterns will also be similar

    • ]]>     2023-03-02 19:07:58 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501602867     PCR aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501604373
  • Polymerase Chain Reaction
  • Used to make MANY copies of a DNA region to study
  • 3 main steps:
    • Denaturation
    • Annealing
    • Extension
  • PCR lets us take a smallsection of DNA and make hundreds of copies
  • This small section can be amplified and examined through many processes
    • For example, gel electrophoresis

  • Commonly used in 

    • ]]>     2023-03-02 19:08:59 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501604373     PCR Step One aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501604685
  • Denuturation 
    • Heatthe strands of DNA to break (denature) them apart into two separate strands
    • This gives room to replicate the desiredgene


    • ]]>     2023-03-02 19:09:13 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501604685     PCR Step Two aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501604980
  • Annearling 
    • Cool DNA so primers can bind
    • Primers bind to areas of the DNA that we want to amplify
      • Two primers needed for each side of DNA


    • ]]>     2023-03-02 19:09:27 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501604980     PCR Step Three aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501605461
  • Extension
    • Heat up DNA again to use taqPolymerase
    • Taq Polymerase adds nucleotidesto extend the primers and replicate DNA
      • This enzyme is adapted to work in high temps!


    • ]]>     2023-03-02 19:09:43 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2501605461     CRISPR aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502627872
  • An enzyme called Cas9(produced by the CRISPR system) will bind to the matching DNA using a “spacer”
    • Spacer: Short RNA sequences that guide the CRISPR system to the target DNA
  • Cas9 will cut the DNA, shutting the targeted gene off
    • Research suggests CRISPR-Cas9 can be used to target and modify “typos” (aka mutations) in the human genome
  • There are other enzymes than Cas9 used in a CRISPR system
    • Ex. - Cpf1
  • PROS
    • Scientists can accerlerate research
    • Rapid diagnostic
    • Correct mutations
  • CONS
    • Ethics (how far is too far?)
      • Potentially wiping out species, creating bacterial resitance


    • ]]>     2023-03-03 13:37:32 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502627872     ELISA aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502630133
  • Enzyme-Linked Immunosorbent Assay
  • Measures antibodies, antigens, proteins, and glycoproteins in a biological sample
  • Diagnosis, pregnancy tests, test for contamination, allergens, disease progression
    • ELISA - INDIRECT
    • Step 1: antibody Coating
      • Specific capture antibody is immobilized on high protein-binding plates
    • Step 2: Capture
      • Samples and standard dilutions are added to wells and will be captured by bound antibodies
    • Step 3: Det4ectionAntibody
      • Specific detection antibody is added to enable detection of captured protein
        • If protein is absent, this antibody will attach to nothing
    • Step 4: Detection conjugated(enzyme)

    • Binds to detection antibody (like a sign)

    ]]>     2023-03-03 13:39:26 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502630133     Western Blot aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502634796
  • Also known at Immunoblot
  • proteins
    • Detect protein of interest
    • Size of proteins
    • Amount of protein expressed
  • We’re using a charged solution to draw proteins through a gel
  • Proteins are sorted by mass and ability to bind to antiboides
    Antibodies are Y-shaped proteins that bind to a specific target (antigen)
    • Used by our immune system!
    • How It Works
    • Proteins in the sample are coated with a detergent to denature (unfold) them AND give them a negative charge
      • We need linear proteins to move through gel
    • Take completed gel & membrane and sandwich between filter paper
    • Negatively charged proteins will be drawn to the positive end
      • The membrane is between the positive end of the chamber and the gel, so the proteins will be drawn onto the membrane
        • They’ll be “blotted” onto the membrane!
        • This membrane is an identical copy of the gel
    • Why the membrane?
      • Gel is too fragile for next steps
      • Antibodies could become stuck in the gel and give false results
    • Once proteins have moved onto the membrane, we add antibodies to locate the proteins we want to see
      • The antibodies act like a flag on the target protein
    • Once the membrane copies the gel, it’s moved to a chamber and exposed to primary antibodies
      • This primary antibody specifically attaches to the protein we’re looking for
      • Once attached, the membrane is washedso extra antibodies don’t stick around
    • A secondary antibody is then added to the membrane
      • This secondary antibody attaches to the primary antibody, not the protein
      • Contains a reporter enzyme that will produce light or color
    • The reporter enzyme allows us to locate the proteins!
    • By adding the antibodies, we only see the proteins we’re looking for on the final membrane

    ]]>     2023-03-03 13:43:37 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502634796     Northern Blot aschofield242 https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502635085
  • Very similar process to the Western Blot, but this test looks for RNA, not protein


    • ]]>     2023-03-03 13:43:54 UTC https://padlet.com/aschofield242/u8vitovr5ejf181r/wish/2502635085