Precision Plue Protein Kaleidoscope Prestained Standards Molecular Weight (KD) Distance Migrated (mm) 37 23 25 30 20 35 15 42 51.5 10 Distance Migrated (mm) vs. Precision Plue Protein Kaleidoscope Prestained Standards Molecular Weight (kD) 40 30 Precision Plue Protein Kaleidoscope Prestained Standards Molecular Weight 20 10 25 30 35 40 45 50 Distance Migrated (mm) STUDENT MANUAL POST-LAB ACTIVITY Procedures Generate a standard curve to calculate protein sizes The different protein bands in your gel can be defined by their different molecular masses. Indeed many proteins are named for their molecular weights. For example p53, a protein implicated in tumor progression is 53 KD in size. To determine the molecular masses of the proteins, a standard curve is created plotting the known molecular masses of the proteins in the Precision Plus Protein Kaleidoscope prestained standards against the distance they have migrated down the gel from the base of the well. A 15% polyacrylamide gel is designed to separate small proteins-proteins less than 40 KD. Your gel analysis will concentrate on this size range. Note: If a different percentage acrylamide gel or an agarose gel has been run, analyze the section of the gel that has the best separation 1. As shown in the figure below draw a line between the 37 and 25 KD bands of the prestained standards. Your gel analysis will be restricted to the proteins below this line. Stenders Actin & Myosin 4 Measure distance from base of wells to the base of the bands 250 150 100 75 50 37 25 20 Measure Standard bands between 37 and 10 kD 15 Measure fish protein bands between 30 and 10 KD 10 - Fig. 18. Image of fish muscle proteins separated by SDS-PAGE and stained with Bio-Safe Coomassie stain. Lines illustrate measurement of bands for constructing the standard curve. STUDENT MANUAL POST-LAB ACTIVITY Define the characteristics (proteins) of the different fish 4. For each fish sample that has been analyzed, determine the molecular masses of the proteins below the 25-37 KD line. Measure the distance each band has migrated from the base of its well. Find that distance on the x-axis of the standard curve. Draw a line up from the x-axis to the curve. Read across to the y-axis to determine the molecular mass. Alternatively, use graphing software to generate the standard curve. Make a line of best fit (or trend line) through the points and formulate an equation to calculate the mass of the unknown proteins on the gel. 5. Enter this data into a table with the molecular masses of the proteins for each fish (see example below). Fish Species A Distance Molecular Migrated Mass (mm) (KD) 25 32.5 26.5 31 29 28.6 36 21.7 21.2 36.5 39 18.8 44 13.9 52 6 Determine which fish have each characteristic (protein) 6. Make a table with a row for every band size you have recorded for all your fish samples and a column for each type of fish on your gel. Then make a mark in each cell of the table where the fish has that size band (see example below). -LAB ACTIVITY ENT MANUAL Species A Species B Species c Species D Species E х X х х X х X х X х 27.5 X х х х X х X X X X 33 X х х X X х х X х Characteristic х Protein Distance Molecular Migrated Mass (mm) (kDa) 25 32.5 26 31.5 26.5 31.0 30.0 28.5 29.1 29 28.6 30 27.6 30.5 27.1 32 25.6 24.7 34.5 23.2 35.5 22.2 36 21.7 36.5 21.2 37 20.7 37.5 20.2 38 19.7 38.5 19.3 39 18.8 39.5 18.3 40.5 17.3 41 16.8 41.5 16.3 42 15.8 43 14.8 44 13.9 45 12.9 46 11.9 46.5 11.4 47 10.9 47.5 10.4 51.5 6.5 52 COUNT х х X X X х х х X х х х х х х х X х X X х X 6.0 х 8 10 13 10 12 Find the number of characteristics shared by each of the fish 7. In the table below both the row and column headings are the types of fish. From the table above, separately compare the number of bands (X's) in common with every other fish sample from your gel and put those numbers into the table below, such that each fish is individually compared with every other fish. In this example, species A and B have just 2 bands in common while species B and C have 10 bands in common. Your table will be the basis for drawing your cladogram. Species A Species B Species c Species D Species E Species A 8 2 2 2 Species B 10 10 5 3 Species C 13 5 Species D 2 Species E

Precision Plue Protein Kaleidoscope Prestained Standards Molecular Weight (KD) Distance Migrated (mm).

Procedures Generate a standard curve to calculate protein sizes The different protein bands in your gel can be defined by their different molecular masses. Indeed many proteins are named for their molecular weights. For example p53, a protein implicated in tumor progression is 53 KD in size.  

To determine the molecular masses of the proteins, a standard curve is created plotting the known molecular masses of the proteins in the Precision Plus Protein Kaleidoscope prestained standards against the distance they have migrated down the gel from the base of the well.

Therefore, Precision Plue Protein Kaleidoscope Prestained Standards Molecular Weight (KD) Distance Migrated (mm).

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