LAB - Native Protein PAGE (PolyAcrylamide Gel Electrophoresis)

Picture downloaded from www.bio-rad.com

Today we ran a native protein PAGEs to find out the size (molecular weight in Daltons) of our GFP purifications. We used two pre-stained standards: BIO-RAD's Precision Plus Protein Broad Range and Kaleidoscope standards (the specific sizes of the bands in the stantards coming to this blog soon).

Before staining the gel we took pictures using UV light and a fluorescent ruler, so we can measure how much the GFP bands (one from each one of the different amounts of extract, 5, 10, and 15 μl) migrated from the origin.
Once the gels are stained (to make our proteins visible) we will make a new measurement since it is likely the gel will shrink a little bit. Also, other proteins that happen to be hydrophobic as well may show up.

By comparing the migration of our GFP with the migration of the proteins in the standards we will be able to estimate the size of the GFP.

Ideally we should have used more standards and duplicate or triplicate our samples, but for the purposes of this lab what we did is more than enough.

Gels were dried afer staining, following a protocol in which hey are wrapped in cellophane paper

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LAB - Measuring protein concentration and mass - Bradford Determination

Today in lab we created a standard to measure protein concentration based on the absorbance of different concentrations of the proteins Bovine Serumm Albumin (BSA) and Bovine Gamma Globulin (BGG, technically known as IgG, for Immunoglobulin G) in 1x Bradford dye reagent, or more precisely, an acidic solution of Coomassie® Brilliant Blue G-250 dye, which absorbance maximum shifts from 465 nm to 595 nm when binding to protein occurs.

We added a range of protein concentrations to the dye reagent, and measured absorbance at 595 nm. The more protein there is in a sample the more dye will be shifting absorbance to the wavelengt we used and the more absorbance will be observed. That is the principle of the Bradford method.

Using such standards (BSA abd IgG) we are going to get an idea of the concentration of our problem sanple, GFP. Ideally, we would use a range of GFP concentrations to create a standard and make an accurate measurement, but let's pretend we just discovered a new protein, and we know nothing about it. We use known standards to get a rough idea of how much protein we purified.

Tomorrow we will run our GFP sample in a native polyacrylamide gel, measure the distance the band migrates in the gel, compare it with some protein standards, an estimate the molecular weight (size) of the GFP. Then we'll have an estimate of how much "novel" protein we purified, and what size it is.

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Exam 01

The exam took place in the regular classroom at 7:30 a.m. Since everybody was able to finish under an hour next time we will meet at 8:00 a.m., same as in a regular class day.

Come see me in my office to check your scores and make sure they are right.

Answer to question 19 (A codon... may code for one or more amino acids) is wrong, so everybody gets 2 points in addition to their final score (a codon codes only for ONE amino acid, or it works as a stop codon. An amino acid can be coded by more than one codon). That mistake actually boosted the mode from a B to an A. Good job, guys!

Results were good over all. Some stats:

• Mean score: 85.5
• Standard deviation: 8.83
• Mode: 90

Score frequency distribution

Toughest questions (statistically):

26. A conserved gene is...
A gene that accumulates very few mutations

Comment: Every gene has mutated in some way or another. No gene, no matter how conserved it is, is identical accross the tree of life. It may accumulate very, very few mutations, but it has them nevertheless.

27. Mutations will accumulate faster in...
An intron

Comment: Exons and regulatory sequences, specially promoters, have important functions. Most mutations in such sequences will be deleterious. Introns, on the other hand, are free to mutate.

40. Bacteria increase genetic diversity through Horizontal Gene Transfer (HGT). Eukaryotes do it mainly through...
Recombination

Comment: Recombination is what happens after fertilization, and it is when paternal and maternal chromosomes exchange information (recombine). Genetic diversity is thus maintained or increased. There are very few cases of HGT in eukaryotes, and it happens mostly in plants. In prokaryotes HGT is rampant.

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Genome evolution

Fig 4-83 (Alberts et al.). Detection of multispecies conserved sequences

We covered pages 245-253 in the book, the final part of chapter 4, regarding how genomes evolve.

Reading for next week (Monday october 20th): Chapter 5, pages 263-281.

REMINDER
October 20th is close. You must turn in the first draft of the literature review papers...!

Quiz 8 Q&As:

1. What is a mitotic chromosome?
A chromosome in its condensed version, visible with a light microscope during mitosis

2. What is conserved synteny?
The fact that the same genes are found in the same order in the genomes of different species (usually related species)

3. Mention two of the three condensed chromosome regions important for chromosomal integrity and function during mitosis
Telomere, centromere, replication origin

4. What is a polytene chromosome?
Found in secretory cells of some fly larvae, are chromosomes that have replicated many times without separating, so they clump in thick groups. The genes do not slide, so when stained the bands corresponding to particular loci are highy visible

5. A nucleosome is composed by a complex of histones and what else?
DNA

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