B Y V E R O N I C A G A LV E Z A N D V I R G I N I A W H I T T I N GT O N

Confirming Fish Identities using SDS-PAGE

Introduction

  Every fish in the sea has proteins at varying levels   Each fish species has a different diversity and mix of multiple

proteins, unique to each kind of fish.   Many fish have been falsely advertised as a species which it is not.   This experiment has been conducted to combat fraudulent fish

marking and advertising at restaurants and grocery stores.   Proteins can be used as a comparative tool

•  We used tested fish from the restaurants Dami, Shines, and Sushi & Sushi

SDS-PAGE •  Protiens need to have

an overall negative charge to flow through the gel

•  SDS coats the protiens in a negative charge

•  Fragments separate based on size

•  Larger on top

•  Smaller on the bottom

•  Each well holds a different sample

Data Analysis

  With the Salmon gel, results were varied in protein content, enough to raise questions about its identity.

  Some of the restaurants tested had only about half the bands they needed to have in order to match the control, in the salmon section of the experiment.

  In the Tuna gel all three restaurants had a protein at the bottom of the gel that the control lacked.

  We concluded that because every other aspect of their protein content was identical, we could consider the tuna to be a match.

  Hamachi and Tuna both were matches to the control

Research Question: Are the fish one buys at a grocery store and restaurant the fish

they are advertised to be?

Hypothesis: Some of the fish at the grocery store and restaurant are not what

they are advertised to be?

Procedures: We will use a standard ladder to compare for base pair counts. Our control will be three different fish from New Sagaya. Our variables will be the same

fish, three from Dami, Shines, and Sushi & Sushi.

Protocol: Label 12 1.5 ml microcentrifuge tubes with the names of the fish.

Add 250 microliters of laemmli buffer to each tube Cut a 2mm piece from each fish sample and transfer into the corresponding

tube. Flick the tube to agitate. Incubate for 5 min at room temp

Label 12 microcentrifuge tubes with the names of the fish above Carefully transfer the buffer from each tube into the new corresponding tube

by pouring. Do not transfer the fish Heat the protein extracts in the tube for 5 min at 95 degrees C.

Attain a Mini-PROTEAN TGX gel. Pull the tape off the bottom of the gel and remove the comb

Clamp the gel into the electrode assembly with the short plate facing inward. Clamp another gel to the opposite side. Place the electrode

assembly into the gel tank, matching the red electrode to the red side of the tank

Fill the inner chamber of the electrode assembly completely with 1xTGS. Pour 1xTGS to the proper fill line of the outside chamber. Load the standard

ladder into both gels Heat the fish samples to 95 degrees C for 2 min

Record the loading order of the gels Load 5 microliters of the standard ladder and 10 microliters of each fish

sample Place the lid on the chamber. Connect the power supplies, red-to-red,

black-to-black. Turn on the power and run the gel for 200 V for 30 min.

When the run is done, turn the power off and remove the lid. Remove the electrode assembly and pour the buffer from the inner chamber back into

the tank. Remove the gel cassette from the electrode assembly. Remove the gel from the cassette by the opening key. Line up the arrows

on the opening lever with the four arrows on the cassette to open. Very gently pick up the gel and transfer into a gel-staining tray. Handle from

the bottom Rinse the gels in tap water in the gel-staining tray. Wash three times for 5

min Pour off the water and add 50 ml of Bio-Safe Coomassie stain. Stain two

gels per staining tray. Stain for one hour with gentle shaking Discard the stain and rinse the gels two times. De-stain the gels in a large

volume of water overnight. Take a photo while the gel is still wet. Dry the gel between two sheets of

cellophane. Using a ruler cut the bottom of the raised edge off the gel using a downward

chopping motion Wet two pieces of cellophane thoroughly with tap water. Stretch the

cellophane over the frame so that it is tight and flat. Place the gel onto the wet cellophane. Pour water over the gel

Carefully place the next sheet of cellophane over the gel, no air bubbles. Add the second rubber band

Set aside to dry

Bibliography: •  FishWatch. (n.d.). NOAA. Retrieved April 10,

2014, from http://www.fishwatch.gov/buying_seafood/identifying_seafood_fraud.htm

•  Foulke, J. E. (1993). Cracking down on fresh

fish fraud. Consumers' Research Magazine, 76(10), 25.

•  Foulke, J. E., & Sudduth, M. (1993). Is something fishy going on? FDA Consumer, 27(7), 12.

•  FAO Fisheries & Aquaculture - Proteins. (n.d.). FAO Fisheries & Aquaculture - Proteins. Retrieved April 15, 2014, from http://www.fao.org/fishery/topic/14869/en

The Hamachi, Salmon, and Tuna Gels

Results:

With this experiment we wanted to ensure that the fish you order at restaurants is the fish it was advertised to be. We did this by extracting the protein from three different fish: Hamachi, Tuna, and Salmon. We got each of these three fish from three different restaurants: Dami, Sushi & Sushi, and Shine's Sushi. With the Salmon gel, results were varied in protein content, enough to raise questions about its identity. Some of the restaurants tested had only about half the bands they needed to have in order to match the control, in the salmon section of the experiment. In the Tuna gel all three restaurants had a protein at the bottom of the gel that the control lacked. We concluded that because every other aspect of their protein content was identical, we could consider the tuna to be a match.

Abstract: In our experiment we have concluded that all the fish from Dami, sushi & sushi, and shines are indeed the correct fish and none are fraudulent. However, there is a clear difference in the sources you want to be buying your fish from. Even though we concluded that the fish were all indeed their respective speciesHamachi and Tuna were the closest to the New Sagaya control. Salmon did match but only to a certain extent. Many things could have occurred to allow for this discrepancy, such as farm raised versus wild and what the fish had been eating.

Conclusion:

This experiment was conducted to combat fish fraud. We have concluded that all Hamachi and Tuna samples match the control enough to be considered the same and therefore, are the fish they are advertised to be. For our Salmon section of the experiment, only the restaurant Shine's Sushi’s samples, matched the control. For the other two restaurants, Dami and Sushi&Sushi, the results were inconclusively different. There are many factors that could have caused this discrepancy in protein levels, such as wild versus farm-raised in source, diet of the fish, freshness, or identity of the fish overall.

Hamachi Gel:

•  Lane 1: Standard

•  Lane 2 & 3: Control Source: New Sagaya(10ul) & (5ul)

•  Lane 4 & 5: Dami: Source 1 (10ul) & (5ul) •  Lane 6 & 7: Shines: Source 2 (10ul) & (5ul) •  Lane 8 & 9: Sushi & Sushi: Source 3 (10ul)

& (5ul)

Background: Proteins are made up of chains of amino acids. SDS-PAGE

stands for sodium dodecyl sulfate- Polyacrylamide gel electrophoresis. Any gel is used to observe either proteins or DNA on a side by side, organized by a progression of bands in each sample’s specific lane. From running an SDS-PAGE you can determine diversity in either protein or DNA. SDS-PAGE separates proteins based on their masses, the bigger staying at the top of its gel lane and the smaller on the bottom of its gel lane. Pieces of muscle are submerged in Laemmli buffer to coat them in SDS, which gives the proteins an overall negative charge and denature the proteins. For the proteins to flow down the gel they must have a negative charge, which attracts them to oppositely charged end. In addition to that they must be denatured so that the proteins become linear and flow correctly as well and because each protein has a different mass they will flow and separate in the gel. Next the samples are heated then ran in a gel. According to the Food and Agriculture organization of the United Nations, fish commonly contain structural proteins such as actin, myosin, tropormysonsin, sarcoplasmic proteins, such as myoalbumin, globulin and connective proteins, such as collagen. Because of a study done by Aesociety, we can assume that various fish species have protein diversity due to their running of different fish varieties in the SDS-PAGE fashion. From the results of our gel we can compare the protein diversity of our different fish samples, just like we in class and Aesociety did. From the data from the gel we can determine whether or not the fish have the same protein identities. After many articles realized about seafood fraud people are worried about the fish they are consuming. An article, which only serves to increase concern about fish safety states that, “Mislabeling can even threaten public health. For example, in 2007 several serious illnesses resulted from the illegal importation of toxic puffer fish that had been mislabeled as monkfish to circumvent U.S. import restrictions for this product.” (fishwatch.gov). In our project we want to compare the protein diversity of fish to ensure they are the advertised species. We want to assure consumers that their purchase is credible.

Confirming Fish Identities using SDS-PAGE

9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 10 9 8 7 6 5 4 3 2 1

Salmon Gel:

•  Lane 1: Standard •  Lane 2 & 3: Control Source: New Sagaya (10ul)

& (5ul)

•  Lane 4 & 5: Dami: Source 1 (10ul) & (5ul)

•  Lane 6 & 7: Shines: Source 2 (10ul) & (5ul) •  Lane 8 & 9: Sushi & Sushi: Source 3 (10ul) &

(5ul) & (10ul)

Tuna Gel:

•  Lane 1: Standard •  Lane 2 & 3: Control Source: New Sagaya

(10ul) & (5ul)

•  Lane 4 & 5: Dami: Source 1 (10ul) & (5ul)

•  Lane 6 & 7: Shines: Source 2 (10ul) & (5ul) •  Lane 8 & 9: Sushi & Sushi: Source 3 (10ul) &

(5ul)

The sushi in this photograph is exactly like the sushi used in the experiment. Tuna is on the left. Hamachi is in the middle. Salmon

is on the right.

The Data Tables: Comparing Protein Bands

Hamachi  

New  Sagaya  (mm)  

Dami  (mm)  

Shines  (mm)  

Sushi  &  Sushi  (mm)  

37   x   x   x  42   x   x   x  

48   x   x   x  

49   x   x   x  

50   x   x   x  

52   x   x   x  

55   x   x   |  

Control   Match   Match     Match  

cake

Salmon    

New  Sagaya  (mm)  

Dami  (mm)  

Shines  (mm)  

Sushi  &  Sushi  (mm)  

38   |   x   |  42   x   x   x  43   |   x   |  46   x   x   x  48   |   x   x  50   |   x   |  52   x   x   x  55   |   x   |  

Control  Inconclus

ive   Match   Inconclusive  

Tuna    

New  Sagaya  (mm)  

Dami  (mm)  

Shines  (mm)  

Sushi  &  Sushi  (mm)  

|   53   53   53  

Control   Match   Match   Match