Module 3, Lab 10 - Protein quantitationBradford dtermination of GFP

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Bradford reagent, based on Brilliant Blue G-250) to bind to proteins (causing the dye reagent to change from a reddish-brownish color to blue) and measure its absorbance. The more concentrated the protein it binds, the darker the blue resultant color, and the greater the absorbance at 595 nm.

Two relative standard proteins are used, bovine serum albumin (BSA) and bovine gamma-globulin (BGG), to generate absorbance vs. protein concentration curves and then interpolate the absorbance of problem samples (mostly with GFP) to estimate their concentration. The problem samples were obtained from the Hydrophobic Interaction Chromatography (HIC).

This method is applied when researchers in proteomics discover a new protein and are trying to gather information about it. In our case, we "discovered" GFP, although we wouldn't have a name yet, had it been a truly newly discovered protein.

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Module 3, Lab 09 - Hydrophobic Interaction Chromatography (HIC) of GFP

Friday, October 21, 2010

Today we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacteria was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected should glow.

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

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Module 2, Lab 07 - Sequencing reactions of GAPC gene

After the cloning process of the GAPC gene from Arabidopsis, and extracting the plasmid DNA (to isolate the pJet1.2 plasmid) we did a RED to confirm the success of the ligation.

Using the samples that had the GAPC gene insert we mixed purified plasmid DNA with forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R), and put them in a 96-well plate. The plate will be shipped to the DOE Joint Genome Institute (JGI) to be sequenced as part of their Sequencing Training Program (STR). The results should be in in two weeks, ready to be used in the bioinformatics labs

We will discuss the DNA sequencing technique most commonly used: Dye-terminator sequencing, a modification of Sanger's chain termination sequencing protocol, which allowed the automation of the DNA sequencing process.

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Guest lecture by Dr. Renee Reijo-PeraEarly human embryo development and associated gene expression

We had the privilege of having Dr. Renee Reijo-Pera, the director of the Human Embryonic Stem Cell Research Center at Stanford University, as a guest lecturer in our class. She shared with us her lab's findings in recent years on stem cell research and early human embryo development.

Some of the main topics in her lecture included...

• Maternal vs. embryonic gene expression - Stages at which maternal mRNAs are active, and then degraded, and at which embryonic mRNAs are synthesized
• Dynamics of cell division between fertilization and blastocyst stage
• Prediction, at day 2 of development, of which embryos are viable (will successfully reach blastocyst stage) - Development of an algorithm to make an objective prediction
• Things we do not know about human embryo development and how stem cell research can help
• How embryo images were obtained and made into movies to allow analysis of developmental process
• Analysis of gene expression - analysis of mRNA from 96 selected genes, extracted from a single cell
• How the development process is correlated with patterns of gene expression

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Lecture, chapter 11 - RNA processing+ info about guest lecturer, Dr. Renee Reijo-Pera

Today we finished chapter 11 on RNA processing. We focused mainly on alternative splicing and how it produces different mRNA molecules by transcribing the same gene.

We also discussed processes like base modification, base substitution, RNA editing, and RNA degradation.

After finishing the chapter we discussed students' impressions on Dr. Reijo-Pera's Keiser lecture yesterday evening and expectations for her talk in our class tomorrow...!!! (expectations from the talk itself and about students' interaction with Dr. Reijo-Pera)

Tomorrow:

Dr. Renee Reijo-Pera, from Stanford University, will give a lecture on human preimplantation development and gene expression and pathways during the first few days of development. We will be joined by students in Dr. Aulthouse's Developmental Anatomy class, and potentially Dr. Walden's CLS program so the room will be packed. The talk will be as exciting as the Keiser lecture and having two-three classes in the audience will make the discussion more interesting and lively...!

Students should be ready to ask questions to, and engage in a discussion with, Dr. Reijo-Pera. Please check the following links:

• The Reijo-Pera lab
• People at the Reijo-Pera lab
• Dr. Reijo-Pera's publications
• Dr. Reijo-Pera's contact info

It will be an exciting day. Take advantage of it!

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