determining protein molecular weight and purification of green and blue florescent proteins

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Question description

I have to write a lab report 6 pages minimum about determining protein molecular weight and purification of green and blue florescent proteins. the lab report should be in 5 different sections, which are objective, background, results, discussion, and answer given questions. on the result section, we need to conduct scatter linear graph.

I am attaching the instruction page as well as the ppt and all the results I got.

 determining protein molecular weight and purification of green and blue florescent proteins
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 determining protein molecular weight and purification of green and blue florescent proteins
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 determining protein molecular weight and purification of green and blue florescent proteins
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BCHB 507/508 Laboratory Applications of Biotechnology Session 15 Lab 153: Polyacrylamide gel Electrophoresis: Determining Protein Molecular Weight Lab 999: Real Time PCR Demonstration Lab 255: Purification & Size Determination of Green & Blue Fluorescent Proteins BCHB 507/508 Laboratory Applications of Biotechnology Lab 153: Polyacrylamide gel Electrophoresis: Determining Protein Molecular Weight Determination of Protein Molecular Weight  Direct Calculation: Must know amino acid sequence.  Does  not take into account modifications. SEC: Size Exclusion Chromatography.  Calibrate column with standards and measure sample retention time.  PAGE:  Does not require expensive equipment.  Native gel vs. SDS gel.  Denature and bind SDS (net negative charge on protein).  Run on gel and compare to standards. SDS-PAGE    SDS-PAGE = Sodium DodecylSulphatePolyAcrylamide Gel Electrophoresis) SDS-PAGE separates proteins according to their size Need to understand: SDS and PAGE meanings SDS = Sodium DodecylSulfate  SDS = Sodium DodecylSulfate anionic detergent  denatures secondary and non–disulfide–linked tertiary structures  SDS confers a net negative charge to the polypeptide in proportion to its length   Polypeptides become rods of negative charges with equal charge per unit length. Polyacrylamide Gels     Have smaller pores than agarose, therefore high degree of resolving power Polyacrylamide gel electrophoresis separates proteins based on Size (Molecular Weight) The gel (matrix) composed polyacrylamide Acrylamide monomers polymerize into long chains that are covalently linked by a crosslinker  Acrylamide is a potent neurotoxin and should be handled with care! Polymerization of acrylamide TEMED - crosslinking agent (19:1 ratio of acrylamide to bis maximizes crosslinking) Higher % of gel smaller pores (holes) so smaller fragments can be resolved http://nptel.ac.in/courses/102103047/module3/lec13/5.html PAGE = PolyAcrylamide Gel Electrophoresis   PAGE is the preferred method for separation of proteins Movement of Proteins in Gel: smaller proteins will move through the gel faster while larger proteins move at a slower pace http://www.biologyexams4u.com/2014_01_01_archive.html SDS-PAGE  Protein sample treated with SDS and beta-mercaptoethanol    SDS disrupts secondary and tertiary structure The mercaptoethanol reduces disulfide bonds Results of treatment: All proteins contain only primary structure and  All proteins have a large negative charge   They migrate through a gel towards the positive pole at a rate proportional to their linear size  Molecular weights with respect to size markers may then be determined http://www.biologyexams4u.com/2014_01_01_archive.html Protein gel electrophoresis Determination MW of Unknown Proteins www.edvotek.com/153.pdf Running SDS-PAGE 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Unsolidified gel (or Acrylamide crystals) are toxic, handle gel with gloves. Remove gel from the pouch and remove tape. Rinse the gel with DI water. Carefully remove the comb and insert gel into chamber (check orientation). Add buffer to chamber (make sure the wells are covered). Use a transfer pipet to rinse out the wells with buffer (often there is unpolymerized material in the well). Boil samples for 3-4 minutes and quickly load samples into wells. (Set tip slightly into well and expel sample.) Set cover on gel box and electrophoresis at 125 volts for 60 minutes (watch for the dye front to reach the bottom of the gel). Turn off the power supply, drain and rinse the gel box and remove the gel cassette from the gel box. Set the gel cassette down with the smaller plate up. Using a spatula crack the edges of the cassette to separate the top and bottom plates. Gently lift and separate the smaller plate from the larger plate, the gel should remain on the larger plate. CAUTION: The gel is very thin and can tear. Prepare the tray with the fixing solution and set the plate with the gel into the solution. Transfer the gel to a tray of staining solution and float a sheet of gel Stain in the solution. Allow the gel to stain for 1-3 hours (if required staining can proceed overnight). BCHB 507/508 Laboratory Applications of Biotechnology Lab 255: Purification & Size Determination of Green & Blue Fluorescent Proteins What are Green and Fluorescence Proteins and Why study them?    Discovered from Aequorea Victoria Widely expressed in E. coli Visual markers (“reporter molecule”) for various studies      Localization and regulation of gene expression Cell movement Protein-protein interactions Screenable marker to identify transgenic organisms Several variants: blue, red, ect. fluorescent proteins https://en.wikipedia.org/wiki/Green_fluorescent_protein GFP derivatives Column Chromatography  Chromatography used for protein purification  Size exclusion  Affinity  Ion exchange (Lab 302)  Hydrophobic interaction (today) cellularphysiology.wikispaces.com Hydrophobic Interaction Chromatography (HIC) = Salting Out    Add bacterial lysate to column matrix in high salt buffer Wash less hydrophobic from column with low salt buffer Elute GFP from column by adding a no-salt buffer Column Chromatography  Packing and Equilibrating the column • • •  Collecting Column Fractions of (gfp) Proteins      Label eight tubes # 1-8 Load the column with gfp extract Add 1X Elution buffer to column & collect 0.5 ml fractions in labeled tubes Store on ice Collecting Column Fractions of (bfp) Proteins       Mount column on ring stand STRAIGHT. Mix and load matrix (avoid bubbles). Wash packed column. Wash column Label eight tubes # 9-16 Load the column with gfp extract Add 1X Elution buffer to column & collect 0.5 ml fractions in labeled tubes Store on ice Identifying the tubes with the highest fluorescence of (gfp) and (bfp) using long wave U.V. light www.edvotek.com/302.pdf HIC vs. SDS-PAGE SDS-PAGE     An analytical technique used to detect the presence of a protein of interest Separates molecules based on size HIC   Can compare denatured and intact proteins to study protein structure  High concentration of GFP from whole cell lysate samples (dense colonies)  Used to isolate a protein from a complex mixture of molecules based on its physical and/or chemical properties Separates molecules based on hydrophobicity Need to lyse open the cells to run the soluble proteins over the column Very dilute concentration of GFP in the HIC column fractions Resources  Packing a chromatography column  https://youtu.be/G4jyd8L0MWE  Performing chromatography  https://youtu.be/VP6Px8zTDNM  Performing SDS-PAGE  https://youtu.be/eaETFKXtNRA  Real Time PCR animation  https://www.youtube.com/watch?v=EaGH1eKfvC0

Tutor Answer

Chucks574
School: UC Berkeley

Thank you so much

Running head: Lab report

1

Lab report:
Name:
Institution affiliation:
Date:

Lab report

2

Objectives:
The objectives of this experiment are to improve our understanding in relation to how
SDS-PAGE works with proteins so as to be able to determine the molecular weight of protein.
Additionally, in this experiment we will investigate the Purification as well as Size
Determination of Blue & Green Fluorescent Proteins.
Background:
In biochemistry, SDS- PAGE in is very essential. This is so since it enables scientists to
be able to separate proteins by their size. This is essential since, whenever bacteria is being
utilized to come up with proteins which are medicinally useful by transformation, then protein
which is of interest should be separated from other cellular proteins. One advantage SDS- PAGE
electrophoresis has over other methods of determining molecular weight of protein is that, it does
not require expensive equipment. SDS- PAGE comprise of 2 essential parts. This are Sodium
Dodecyl Sulfate (SDS) & Polyacrylamide Gel Electrophoresis (PAGE).
SDS is an anionic detergent with negative molecules which can dissolve molecules which
are hydrophobic in nature. SDS usually denatures secondary as well as non-disulfide- linked
tertiary structures. Whenever Proteins are dissolved in SDS, their structure changes from their
usual coiled up secondary, quaternary structure on to a primary structure which is usually flat &
covered with...

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Anonymous
Excellent job

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