Fish Protein/ Proteomics Rubric Protein Fish Lab Quick Guide Introduction to protein gels  Post Lab Analysis Help (30 points)

Group 1

Group 2

Group 3

Group 4

chicken

beef

chicken

beef

tuna

snapper

tuna

snapper

sole

cod

cod

sole

salmon

salmon

salmon

salmon

trout

trout

trout

trout

Title: (1 point)

The title is a statement (not a question) reflecting the independent and dependent variables. Example: The Effect of Various Concentrations of Sucrose on Carrot Slices.

Results/Trends:

1) Standard Semi-Log Graph of mm migrated vs. kDa protein of Kaleidoscope Standard: graph should show use to determine kDa sizes of fish unknown bands and Actin/Myosin control . Graph can either be on semi-log paper or computer generated.(3 points)

2) Picture of gel, Data Table of gel lanes with mm migrated and kDa sizes, contains all  organism samples used in the lab, see  Post Lab Analysis Help for example on page 5) (6 points)

3) Make a data table of number of bands in common (see Post Lab Analysis Help) (3 points)

4) Make the cladogram (see  Post Lab Analysis Help) of the  species examined during the lab.(3 points)

5) Post Lab Analysis Help & complete data table of amino acids and cladogram of the supplied information.(4 points)

6) HBB FASTA list using the techniques from  the Whales Activity (here is a direct link to the Whales (seals) protein tutorial)

(make sure you copy and paste (or use prtscn) a copy of your tree.Don't forget to look through the  *** at the end of this document for additional help (3 points)

Reflection: Analysis/Conclusion/Lab Review Questions:(5 points)

In general the requirements of this section of the report is to provide an explanation of "why" of the results/data section supports or doesn't support your hypotheses. What information in the data section and the introduction help you explain the results you found? This might include the answers to the questions at the end of an activity.

 References  (Remember that info on how to format references can be found in www.bibme.org) (2 points)

***Explanation of how to use the clustal distance matrix and tree information; (thank you to Mark Miller at NGWB)

Here is a simple example:

The matrix is a pairwise comparison between each of the sequences. The number of mismatches divided by total sequence length gives the Matrix value.

Here each sequence has 22 or 23 characters.

Differences between A and B = 3/22 =0.136 ignore the dash Differences between A and C = 5/22 =0.227 ignore the dash Differences between A and D = 11/23 =0.476 count the dash Differences between A and E = 19/22 =0.864 ignore the dash Differences between B and C = 7/22 =0.318 ignore the dash

Etc....

A -GARFIELDMADCATLIKETHAT

B -GARFIELDBADCATLIKETHIS

A -GARFIELDMADCATLIKETHAT

C -GARFIELDFATRATTAKETHAT

A -GARFIELDMADCATLIKETHAT

D WHATEVER-FATRATTAKETHAT

A -GARFIELDMADCATLIKETHAT

E -THISISADISTINCTPEPTIDE

B -GARFIELDBADCATLIKETHIS

C -GARFIELDFATRATTAKETHAT

 

Based on the alignment we can set up the DISTANCE matrix file:

 

 

"A"

 "B"

 "C"

 "D"

"E"

"A"

 0

0.136

 0.227

0.476

 0.864

"B"

0.136

0

0.318

0.571

0.864

"C"

 0.227

0.318

0

0.238

0.773

"D"

0.476

 0.571

 0.238

 0

 0.857

"E" 

 0.864

0.864

0.773

 0.857

 0

 

The relationship between the numbers in the trees, and the numbers In the matrix?

 

 Cow

  Minke

Dog

HSeal

   RKang

Cow

0.000

0.131

0.179

 0.159

  0.290

Minke_Whale

 0.131

0.000

 0.137

 0.116

 0.247

Dog

 0.179

0.137

 0.000

 0.068

 0.240

Harbor_seal

0.159

 0.116

 0.068

 0.000

 0.226

Red_kangaroo

0.290

 0.247

0.240

0.226

0.000

The Newick tree:

(

(

Cow:0.08678,

Minke_Whale:0.04425)

:0.02602,

(

Dog:0.04283,

Harbor_seal:0.02567)

:0.02205,

Red_kangaroo:0.17658);

CLustalW uses Neighbor joining to get

Break the distance from cow to minke into a node that is 0.08678 from cow + 0.04425 from minke whale  = 0.131 total distance Etc.

Also (from Mark Miller at NGWB):

The 0.0 (listed on the bottom of the phylogenetic trees) is surely a bug in the software (someone else’s that I incorporated). My guess is that these distances are so small that the scale bar is designed to read nothing smaller than one decimal place, and the distance represented there is actually 0.049 so it shows as 0.0. The actual distances can be read from directly the Newick file .

 Another option is to use a very cool interactive tool called phylowidget. http://www.phylowidget.org/

If you visit that site, click phylowidget full, it will open in your browser.

Paste the Newick tree into the box to the right of the main screen that says Tree:

Replace the statement “Paste your Newick formatted tree here:”

With this:

(Red_kangaroo:0.17658,(Cow:0.08678,Minke_Whale:0.04425):0.02602,(Dog:0.04283,Harbor_seal:0.02567):0.02205);

Click in the main box:

The tree will appear.

Now click “View” and in the dropdown, find and check the box branchlengths.

Use your mouse to hover over each node, and the branch length will appear in the box to the right labeled

Node info”

The relationship between all the species is really easy to see in the unrooted view (select View, Layout, Unrooted in the dropdown menu)

Also, this tool allows one to export pdfs as well. Look under “file” and select “export image”

Anyway, that all may be tmi, but this is a nice tool for playing interactively with representations of phylogenetic trees….