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 _
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.
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. respectively. (A kilodalton is 1000 daltons, the units in which molecular weight is measured.)
B com arin arin the ositio osition n of the bands 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:
A B
Protein A size
is about 70 kilodaltons in
Protein B size
is about 60 kilodaltons in
+
SDS-PAGE
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 as een run as n t e prev prev ous example.
A B
C D
Biology 171L, Fall 2000
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.
A B
C D
By comparing the position of the ands 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.
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Separation and Identification of Biological Compounds
SDS-PAGE
SDS-PAGE
If you said “3”, you’re right!
Here’s 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.
1 2 3
How many proteins do you think are in the sample that was run in lane 2?
SDS-PAGE
SDS-PAGE
How many proteins are represented in lane 3?
There are 2 proteins in lane 3. 1
2
What is the estimated molecular weight of the protein(s) in lane 3?
By comparing their position with the known MW of proteins in lane 1, their estimated MWs are 135 and 66 kilodaltons, respectively.
SDS-PAGE But what if you wanted to be more precise than just estimating the molecular weights of the proteins in lanes 2 and 3?
Then you would have to compute their Rf values, and compare those values to a standard curve.
Biology 171L, Fall 2000
We’ll take things one step at a time.
We’ll learn what an R f value is, and how to compute it.
We’ll learn about standard curves.
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Separation and Identification of Biological Compounds
Computing Rf Values
Computing Rf Values Let’s say we are interested in calculating Rf for protein X in lane 2.
Rf stands for “retardation factor”. For each protein, it is calculated as: A
Then A = distance protein X has traveled from top of gel, and B
distance of dye front from top of running gel
= stance o ye ront from top of running gel. A So, Rf = B
An example follows. Protein X
Computing Rf Values
Computing Rf Values
In the original gel, A
A = 11 mm, and B B
= 53 mm A
So, Rf =
11 mm =
B
53 mm
Rf = 0.21
How about ano er example?
Protein X
Computing Rf Values
Computing R f Values
Let’s calculate Rf for protein Y in lane 3. C = distance protein Y has traveled from top of running gel
C
B
B = distance dye front has traveled from top of running gel C So, Rf = B
Protein Y
Biology 171L, Fall 2000
In the original gel, C = 21 mm, and C B = 53 mm B
So, Rf =
= B
21 53
Rf = 0.40
Protein Y
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Separation and Identification of Biological Compounds
Standard Curves Let’s 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:
Standard Curves
Gray Snapper Catch 8 7 6
A standard standard curve is exactly what it is named to be. It is a curve --usually a line-- constructed from 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.
) b 5 l ( t h 4 g i e 3 W 2 1 0 0
5
10
15
20
25
30
Length (inches)
An example example follows.
Standard Curves
Standard Curves
On the way back to shore, you catch another fish, but in the excitement excitement of t he 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
Let’s say the last fish you caught was 22 inches long. To 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
8
) b 5 l ( t h 4 g i e W3
7 6
) b 5 l ( t h 4 g i e 3 W
2
2
1
1
0
0 0
5
10
15
20
25
30
0
Details follow
5
10
Standard Curves A good estimate for the weight of your fish is 5.5 lbs. lbs .
15
20
25
30
Length (inches)
Length (inches)
Standard Curves The best-fit line that you drew through your plotted fish data is
5.5 lbs
called a
“standard curve”.
8
8
7
7
6
6
) b 5 l ( t h 4 g i e W3
) b 5 l ( t h 4 g i e W3
2
2
1
1
0
0 0
5
10
15
20
Length (inches)
Biology 171L, Fall 2000
25
30
0
5
10
15
20
25
30
Length (inches)
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Separation and Identification of Biological Compounds
Computing Rf Values, and Standard Curves
Computing R f Values, and Standard Curves To construct this standard curve: 1
In today’s lab, you will construct a standard curve, not from from fish fish data data but but from from the the sample with 5 proteins of known molecular weight in lane #1 of a gel your Teaching Assistant will assign to you.
2
3
Here’s a general view of what this standard curve will look like:
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 Draw a best best-f -fit it line line
Computing R f 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 . The unlabeled arrows on the gel indicate the proteins in
lane 2 for which you should compute the R f .
Computing Rf Values, and Standard Curves
The arrows labeled “3” indicate the proteins in
Computing R f Values, and Standard Curves Here’s a gel we haven’t 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 ).
lane 3 for which you should compute the R f . A 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.
Biology 171L, Fall 2000
B C
(When a rotein band is quite thick, as in B , C and D , make your measurements from the middle of the band.)
D
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Separation and Identification of Biological Compounds
Computing Rf Values, and Standard Curves
Last, determine the molecular weight for each protein by comparison with the standard curve:
The Rf of each indicated protein was computed as previously described:
Rf . B
0.43
C
0.54
D
0.91
A
B
C
D Details follow
MW A 140,000
B 60,000 C 44,000
D 17,000
A
Biology 171L, Fall 2000
B
C
D
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