Cell Biology Lab 5

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Questions and Answers

What is the primary role of 2-mercaptoethanol in the SDS-PAGE process?

  • To enhance protein color during staining
  • To stabilize protein structures
  • To increase the migration speed of proteins
  • To reduce disulfide bonds and unfold proteins (correct)

In SDS-PAGE, what is the composition of the loading buffer?

  • Glycine, water, and bromophenol blue
  • SDS, Tris, and water
  • Tris-HCl, SDS, glycerol, beta-mercaptoethanol, bromophenol blue (correct)
  • Tris-HCl, SDS, and glycine

How do proteins migrate during SDS-PAGE?

  • Based on their shape and color
  • Based on their molecular charge only
  • Based on their molecular weight and charge
  • Based on their size, moving from negative to positive pole (correct)

Which of the following components is found in the running buffer for SDS-PAGE?

<p>Tris, glycine, and SDS (D)</p> Signup and view all the answers

What is the purpose of the stacking gel in SDS-PAGE?

<p>To allow all protein samples to enter the separating gel simultaneously (D)</p> Signup and view all the answers

What determines the migration distance of the protein bands in SDS-PAGE?

<p>The size of the proteins (A)</p> Signup and view all the answers

Which step follows the loading of samples into the SDS-PAGE gel?

<p>Running the gel at 100V (B)</p> Signup and view all the answers

Which statement about the relationship between Rf value and protein size is correct?

<p>Lower Rf values correspond to larger proteins (B)</p> Signup and view all the answers

What is the primary mechanism by which SDS-PAGE separates proteins?

<p>Based on protein size (C)</p> Signup and view all the answers

Which component of the polyacrylamide gel acts as a cross-linking agent?

<p>N,N'-methylene-bis-acrylamide (Bis) (A)</p> Signup and view all the answers

When the pore size of the polyacrylamide gel increases, what is the effect on protein migration rate?

<p>Migration rate increases (C)</p> Signup and view all the answers

Which step is performed first in a two-dimensional gel electrophoresis?

<p>Isoelectric focusing separation (A)</p> Signup and view all the answers

What role does sodium dodecyl sulfate (SDS) play in the SDS-PAGE process?

<p>It unfolds and denatures proteins (B)</p> Signup and view all the answers

What does the variable %T represent in the context of polyacrylamide gels?

<p>Total concentration of acrylamide and bis-acrylamide (C)</p> Signup and view all the answers

Which of the following components is NOT involved in the polymerization of polyacrylamide gel?

<p>Sodium dodecyl sulfate (A)</p> Signup and view all the answers

Which parameter affects the pore size of the polyacrylamide gel according to %C?

<p>Concentration of crosslinking agent (D)</p> Signup and view all the answers

what are the components of polyacrylamide gel?

<p>Acrylamide: matrix material N’N’-methylene-bis-acrylamide, known as “Bis”: cross-linking agent TEMED: catalyst Ammonium persulfate: initiator (A)</p> Signup and view all the answers

what is polyacrylamide gel

<p>The gel is made by the polymerization of acrylamide and bis, which forms a network of pores. (A)</p> Signup and view all the answers

SDS facts

<p>difference in MW, many proteins can be analyzed bc of this, proteins have unique charges, so tx w SDS will denature it (unfolds it), SDS binds to it and cover protein, SDS is negatively charged, making all proteins similar in charge is goal so it is separated only by MW.... also treat with B-M to reduce disulfide bonds, the poly acrylamide gel has a stacking gel (top, low ph) and separating gel (bottom, high ph), top is cathode (-) and bottom is anode (+), large proteins top and smaller at bottom, then stained with coomassie blue to see band results, first well for marker proteins in kDa, (A)</p> Signup and view all the answers

stacking gel:

<p>Found at the top of the gel and contains the wells. Has lower %T (larger pore size), lower pH. All of the protein samples line up and enter the separating gel at exactly the same time (A)</p> Signup and view all the answers

Separating (a.k.a. resolving) gel:

<p>Has higher %T (smaller pore size), higher pH. Proteins will separate based on size. (B)</p> Signup and view all the answers

Loading (a.k.a. dissociation) buffer:

<p>Tris-HCl, SDS, glycerol, beta-mercaptoethanol, bromophenol blue. (B)</p> Signup and view all the answers

Running buffer:

<p>Tris, glycine, and SDS, pH 8.3. (A)</p> Signup and view all the answers

Rf=

<p>Migration distance of protein from origin (bottom of well)/ Distance the dye front travelled from the origin (A)</p> Signup and view all the answers

kDa

<p>kilodalton (B)</p> Signup and view all the answers

%T and %C

<p>%T= Total monomer concentration %C= Weight percentage of crosslinker (A)</p> Signup and view all the answers

%T

<p>total percentage of acrylamide (monomer) and bis-acrylamide (cross-linker) per 100 ml of solution. (A)</p> Signup and view all the answers

components of polyacrylamide gel:

<p>The pore size determines the rate of protein migration through the gel The pore size is expressed as %T The pore size decreases with increased %T (A)</p> Signup and view all the answers

Flashcards

SDS-PAGE (SDS-polyacrylamide gel electrophoresis)

A technique used to separate proteins based on their size.

Protein Gel Electrophoresis

A method to separate proteins using an electric field and a gel.

Native Gel (a type of protein gel electrophoresis)

Protein separation based on size, charge, and shape of the protein.

SDS

A detergent that unfolds & denatures proteins, giving them a uniform negative charge.

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Polyacrylamide Gel

A gel made from acrylamide & bis-acrylamide used for protein separation (forms a network of pores)

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Acrylamide

The main component in polyacrylamide gel used to create a matrix.

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Isoelectric Focusing

Protein separation based on their isoelectric point (pI).

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2-Dimensional Gel

Protein separation in two steps, first based on isoelectric point (pI), and then by size (via SDS-PAGE).

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SDS-PAGE

A technique used to separate proteins based on their size.

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2-mercaptoethanol

A reducing agent that breaks disulfide bonds in proteins, unfolding them.

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Separating Gel

Part of the SDS-PAGE gel with smaller pores, separating proteins by size.

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Loading Buffer

Mixture of components (Tris-HCl, SDS, glycerol, beta-mercaptoethanol, bromophenol blue) used with protein samples.

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Rf Value

Ratio of the distance a band migrates to the distance the dye front travels in SDS-PAGE.

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Standard Curve

Graph relating the log of molecular weight of protein markers to their Rf values in SDS-PAGE.

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Stacking Gel

Part of the SDS-PAGE gel with larger pores that aligns protein samples prior to entering separating gel.

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Running Buffer

Buffer solution (Tris, glycine, and SDS) used to carry current through gel in SDS-PAGE.

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Study Notes

BIOL 3120 - Cell Biology Lab - Lab 5: SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

  • Lab Objective: Protein separation using SDS-PAGE
  • Types of Protein Gel Electrophoresis:
    • Native gel: separation based on size, charge, and shape
    • Isoelectric focusing: separation based on isoelectric point
    • SDS-PAGE: separation based on size (focus of this lab)
    • 2-dimensional gel: two-step process, first step based on isoelectric point (isoelectric focusing), second step separation is size based through SDS-PAGE
  • Components of Polyacrylamide Gel:
    • Acrylamide: matrix material
    • N,N'-methylene-bis-acrylamide ("Bis"): cross-linking agent
    • TEMED: catalyst
    • Ammonium persulfate: initiator
    • Gel formation: polymerization of acrylamide and bis creates a network of pores
    • Pore size (%T): determines protein migration rate; decreases with increased %T.
    • %T: total percentage of acrylamide (monomer) and bis-acrylamide (cross-linker) per 100 ml of solution.
    • %C: percentage (weight) of crosslinker to acrylamide and cross-linker (weight ratio)
  • SDS-PAGE Procedure
    • Set up precast gel (following video instructions)
    • Mix 5µL protein sample with 5µL loading buffer
    • Heat the sample at 95-100°C for 5 min.
    • Load sample into well(s).
    • Run the gel at 150V until dye front reaches the bottom.
    • Stain the gel with Coomassie (CBB) stain solution
    • Destain the gel with 100mL destain solution.
  • SDS-PAGE Components:
    • Stacking gel: top of gel, lower %T, larger pore size, lower pH; all proteins line up and enter separating gel at same time.
    • Separating gel: lower %T, smaller pore size, higher pH; proteins separate based on size.
    • Loading buffer: Tris-HCl, SDS, glycerol, β-mercaptoethanol, bromophenol blue
    • Running buffer: Tris, glycine, SDS, pH 8.3.
  • Protein Migration (Rf):
    • Migration distance of protein from origin (bottom of well) to dye front.
    • Standard curves: plot log10 molecular weight vs. Rf for protein markers to determine bands size for unknown protein bands.

Post-Lab Questions and Requirements

  • Calculating band size: use standard curve of known molecular weight to determine unknown bands size.
  • use Rf values for unknown bands in standard curve to determine molecular weight.
  • Lab Report Requirements (Lab 5):
    • Introduction: SDS-PAGE and protein electrophoresis.
    • Materials: list chemicals used and instrument.
    • Methods: experimental procedure steps
    • Results: protein gel analysis (figure)
    • Discussion/Conclusion: improvements, result justification, conclusions.
    • File name: BIOL3120_Section_LastName_FirstName_LabN; email lab report to provided email address.

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