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Separation and Identification of Biological Compounds

SDS-Polyacrylamide Gel Electrophoresis

SDS-PAGE
In the next part of the lab exercises, you will examine a gel that has been prepared by using the technique known as SDS-PAGE.

Three types of protein molecules, here represented as are given a net negative charge by treatment with SDS, a powerful, negatively-charged detergent. (A) They will therefore all migrate towards the anode ( + pole) in an electric field. (B) The three types of molecules will separate according to their molecular weight, with the smaller molecules migrating more quickly towards the anode than the larger molecules.

PAGE stands for polyacrylamide gel electrophoresis.

SDS stands for sodium dodecyl sulfate.

SDS-PAGE _
This is a picture of a finished gel, similar to what you will be given in class during the laboratory exercises. The anode (+ pole) was at the bottom of the gel, so the SDS-treated proteins migrated from the top to the bottom of the gel.

SDS-PAGE
In lane #2, a sample containing proteins of unknown molecular weight was run. The arrows on the right point to the two bands resulting from the electrophoretic separation. The two bands indicate there were 2 proteins in the sample.

( ), In the left-hand column (lane 1), a standard with 5 proteins of known molecular weight was run. Labeled arrows on the left point to the 5 bands resulting from the separation of these proteins in the electric field. Their molecular weights (MW) are 116, 97, 66, 45, and 29 kilodaltons, respectively. (A kilodalton is 1000 daltons, the units in which molecular weight is measured.)

A B

y p g p By comparing the position of the bands of the unknowns to the position of the bands of proteins of known molecular weight in lane 1, we can estimate the MW of the unknowns: Protein A size is about 70 kilodaltons in

Protein B is about 60 kilodaltons in size

SDS-PAGE
Here is an example of a different finished gel. The same standard, with the same 5 proteins of known MW, has been run in lane 1. In lane #2, the same sample with 2 proteins of unknown molecular weight has been run as i the previous h b in th i example. This time, however, an additional sample has been run in lane 3. There are two bands resulting from the electrophoretic separation, indicating there are 2 proteins in this sample.

SDS-PAGE

A B

A B
By comparing the position of the bands of the unknowns in lane 3 with the position of the proteins of known MW in lane 1, we can see that their MW is less than 29 kilodaltons.

C D

C D

Biology 171L, Fall 2000

Separation and Identification of Biological Compounds

SDS-PAGE Heres a third example of a finished gel. The same standard with 5 proteins of known MW has been run in lane #1. Lanes 2 and 3 each have a new sample.

SDS-PAGE

1 2 3

If you said 3, youre right!

How many proteins do you think are in the sample that was run in lane 2?

SDS-PAGE How many proteins are represented in lane 3?

SDS-PAGE There are 2 proteins in lane 3. 1

2 By comparing their position with the known MW of proteins in lane 1, their estimated MWs are 135 and 66 kilodaltons, respectively.

What is the estimated molecular weight of the protein(s) in lane 3?

SDS-PAGE

Well take things one step at a time.


But what if you wanted to be more precise than just estimating the molecular weights of the proteins in lanes 2 and 3?

Well learn what an Rf value is, and how to compute it.

Then you would have to compute their Rf values, and compare those values to a standard curve.

Well learn about standard curves.

Biology 171L, Fall 2000

Separation and Identification of Biological Compounds

Computing Rf Values Rf stands for retardation factor. For each protein, it is calculated as:

Computing Rf Values Lets say we are interested in calculating Rf for protein X in lane 2. A Then A = distance protein X has traveled from top of gel, and B B = di t distance of dye f t f d front from top of running gel. A So, Rf =

distance protein has traveled from top of running gel distance of dye front from top of running gel

An example follows. Protein X

Computing Rf Values In the original gel, A A = 11 mm, and B = 53 mm B A So, Rf = B = 11 mm 53 mm

Computing Rf Values

R = 0.21
f

How about another th example?

Protein X

Computing Rf Values Lets calculate Rf for protein Y in lane 3. C = distance protein Y has traveled from top of running gel B B = distance dye front has traveled from top of running gel C So, Rf = B

Computing Rf Values In the original gel, C = 21 mm, and C B = 53 mm C B So, Rf = B = 21 53

R = 0.40
f

Protein Y

Protein Y

Biology 171L, Fall 2000

Separation and Identification of Biological Compounds

Standard Curves
A standard curve is exactly what it is named to be. It is a curve --usually a line-- constructed from known data, against which to estimate the value of an unknown quantity. It therefore provides a known standard by which to assess something that is not known.

Standard Curves Lets say you go fishing offshore for gray snapper (uku) and catch 11 fish. You measure their length and weight, and plot this data on a graph. The graph below shows your data:
Gray Snapper Catch
8 7 6

Weight (lb)

5 4 3 2 1 0 0 5 10 15 20 25 30

An example follows.

Length (inches)

Standard Curves On the way back to shore, you catch another fish, but in the excitement of the catch, your scale for weighing the fish falls overboard. You can estimate its weight, however, by measuring its length, and comparing its length to a best-fit line drawn through your data for the 11 fish: Gray Snapper Catch
8 7 6

Standard Curves Lets say the last fish you caught was 22 inches long. To estimate its weight, draw a vertical line from 22 inches on the X-axis, up to the best-fit line you drew for the known data points. Then read across to the Y-axis to estimate the weight of your fish.
8 7 6

Weight (lb)

5 4 3 2 1 0

Weight (lb)

5 4 3 2 1 0 0 5 10 15 20 25 30

Details follow

10

15

20

25

30

Length (inches)

Length (inches)

Standard Curves A good estimate for the weight of your fish is 5.5 lbs.

Standard Curves The best-fit line that you drew through your plotted fish data is called a

5.5 lbs

standard curve.
8 7 6

8 7 6

Weight (lb)

5 4 3 2 1 0 0 5 10 15 20 25 30

Weight (lb)

5 4 3 2 1 0 0 5 10 15 20 25 30

Length (inches)

Length (inches)

Biology 171L, Fall 2000

Separation and Identification of Biological Compounds

Computing Rf Values, and Standard Curves

Computing Rf Values, and Standard Curves To construct this standard curve: 1 Compute Rf for each of the 5 proteins in lane 1 Using the semi-log paper in your lab manual, plot each Rf value against the MW of its protein (remember the numbers on the gels are in kilodaltons, i.e., 116 is actually 116,000) Draw a best-fit line

In todays lab, you will construct a standard curve, not from fish data, but from the sample with 5 proteins of known molecular weight in lane #1 of a gel your Teaching Assistant will assign to you.

Heres a general view of what this standard curve will look like:

Computing Rf Values, and Standard Curves The next part of the SDS-PAGE lab exercise instructs you to determine the molecular weights of the indicated unknown proteins on your gel. Each gel has the same standard in lane 1, but different samples in lanes 2 and/or 3. 3 The unlabeled arrows on the gel indicate the proteins in

lane 2 for which you


should compute the Rf.

Computing Rf Values, and Standard Curves The arrows labeled 3 indicate the proteins in

Computing Rf Values, and Standard Curves Heres a gel we havent seen yet. The arrows indicate there are 3 proteins in lane 2 ( A , B , and C ) for which Rf and MW should be determined, and 1 protein in lane 3 ( D ). A ( (When a protein band is p quite thick, as in B , C and D , make your measurements from the middle of the band.)

lane 3 for which you


should compute the Rf.

After calculating the Rf for each indicated protein on your gel, determine the molecular weight of each by referring to your standard curve. A final example follows.

B C

Biology 171L, Fall 2000

Separation and Identification of Biological Compounds

Computing Rf Values, and Standard Curves The Rf of each indicated protein was computed as previously described:

Last, determine the molecular weight for each protein by comparison with the standard curve:

Rf
A B C

0.11 0 11 0.43 0.54 0.91


A B C D Details follow

MW A 140,000

B 60,000 C 44,000

D 17,000

Biology 171L, Fall 2000