Fish Proteomic/Western

Blot

By Grant Akalonu
CHE 447-01
Objective/Introduction

The purpose of this experiment was to study the protein composition of the muscle tissue
of various fish species by using SDS-PAGE to separate and analyze the protein samples. Such
studies are used to draw members of a species to a common ancestor by examining similarities in
physiological composition.

Materials/Methods

Sample Preparation
All DTT (256mg) was added to the 30mL bottle of Laemmli sample buffer and mixed
well.
Cut each fish sample into eight roughly 0.25cm square chunks and put in appropriate
labeled micro-tubes. Add 300L of Laemmli sample buffer to each micro-tube.
Flick the micro-tubes well to agitate the tissue in the sample buffer.
Take 30L of each sample (do not transfer the fish) and standard actin-myosin sample
into new labeled micro-tubes. Heat at 95C for 5 minutes, quick spin before loading.

SDS-PAGE (2 gels per group)

Dilute the 10x Tris-Glycine-SDS electrophoresis buffer to 1x TriSGlycine-SDS

electrophoresis buffer.
Set up the SDS PAGE gels in the running chamber.
For gel 1, load 5 L each sample. This gel after running will be stained with Coomassie
stain solution.
For the second gel, load 8 L each sample. This gel will be used to perform a Western
Blot on the nitrocellulose membrane.
For both gel, load 5 L of Standard Molecular Maker, and 20 L for actin-myosin sample
(positive control)
Run the gels at 180V for around 40 minutes then remove gel 1 from the plastic case stain
over night with Coomassie stain. Then, destain it twice before visualization.

Blotting Proteins to the membrane

The transfer buffer solution was prepared by mixing 700mL of distilled water with 200
mL of ethanol and 100mL of 10x Tris buffer.

All of the materials in this step were wet with buffer solution. A sandwich consisting of
the transferring gel-membrane was made in in the following order. On the black side of
the plastic platform we put one wick-pad layer follow by filter paper, SDS-Page gel,
nitrocellulose membrane, one more filter paper layer and one wick-pad layer on top. The
 platform was closed and inserted into the chamber fill with transfer buffer, and run at
150V for 50 minutes.

Immunodetection

Blocking solution was added to the tray with the membrane for 30 minutes.
The blocking solution was discarded, and 10 mL of secondary antibody was added to the
tray and it was placed on a rocker for 50 minutes. Then it was washed with wash buffer
for 3 minutes, 3 times.
After washing, 10 mL of secondary antibody was added again and put on a rocker for 45
minutes.
Then 10 mL of HRP was added, the tray was placed on a rocker, bands developed after
10 minutes. Then it was washed with distilled water, dried, and analyzed.

Results
Lane Sample

1 Ladder

2 Molecular
Marker

3 Flounder

4 Codfish

5 Tilapia

6 Salmon

7 Colossoma
Bradypomum

8 Actin Myosin

Fig.1. SDS-PAGE result from fish proteomics.

 Log of Molecular Weight Marker vs Rf Values
6

4
Log of Mw

y = -1.8242x + 5.6861
3
R = 0.9934
2

0
0 0.2 0.4 0.6 0.8 1
Rf

Fig.2 Graph of Rf vs Molecular Weight.

Marker Mw Distance of Rf log Mw

migration

1 250000 1 0.185185185 5.397940009

2 150000 1.5 0.277777778 5.176091259

3 100000 2 0.37037037 5

4 75000 2.3 0.425925926 4.875061263

5 50000 2.8 0.518518519 4.698970004

6 37000 3.4 0.62962963 4.568201724

7 25000 3.8 0.703703704 4.397940009

8 20000 4.2 0.777777778 4.301029996

9 15000 4.3 0.796296296 4.176091259

10 10000 5.1 0.944444444 4

Fig 3. Western blot results

Discussion & Conclusion

The various types of protein in each fish sample was determined quantitatively by first
constructing a graph of Rf value vs the log of the molecular weight of the protein. From this
graph, a linear equation was used to determine the molecular weight of each protein present in all
of the fish samples. All of the Rf values for each band on the gel were plugged into the linear
equation shown in the graph on Figure 1. An excel spreadsheet accompanies this report, in which
the molecular weight of the protein of each band is highlighted in red. It is difficult to determine
exactly the type of protein is in each fish sample based solely on the given information about the
molecular weight of various proteins. However, it is clear that there are some similarities among
each species of fish in muscle protein structure.