6.2 Cell Bio Methods II Flashcards
What is SDS page? Describe how to conduct this procedure, the mechanism of it, and what you learn from it.
SDS PAGE aka Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is used to separate proteins on the basis of their size
1) Break all disulfide bonds in protein using a reducing agent such as Beta mercaptoethanol - the purpose of breaking disulfide bonds is to avoid possible problems related to proteins folding up into complex structures
2) Proteins are incubated w/sodium dodecyl sulfate (SDS, which is a negatively charged detergent)
3) the protein mixture is placed on one end of some sort of a gel material like polyacrylamide and an electrical field is placed across the gel.
4) the negatively charged proteins will migrate toward the positively charged electrode at speeds which are directly related to the number of SDS detergent molecules that are stuck to the protein, which in turn is directly related to the size of the protein.
5) After an appropriate period of time, the gel can be removed from the electrical field and processed to visualize the location of proteins.
What is isoelectric focusing? How is it different from SDS PAGE?
Separates proteins using their electrical charge, proteins ability to accept charged ions found in their environment, and their ability to migrate toward or away from an electrode if charged or remain still if uncharged. This approach differs from SDS page in that it can examine the regulation of proteins as well as just their amount in a sample
IEF gel can detect some types of modifications to proteins which generates differences in electrical charge. For example, if a protein is phosphorylated vs if it’s not phosphorylated. Phosphorylation puts negative charges on a protein. Therefore if the protein is activated or inactivated by phosphorylation it will migrate to different positions on an IEF gel.
Describe how IEF gel works:
1) Proteins are placed w/in a loose gel that contains a stationary pH gradient of molecules.
2) If a protein is initially negatively charged, it will move toward the positive electrode and moving toward the positive electrode will bring this protein into areas of increasingly high concentrations of positively charged ions,
3) The positive charged ions will then bind to the protein and start to cancel out it’s negative charge.
4) Eventually when the positive charges are gained from the surrounding solution exactly equal the proteins own negative charge, the protein has a neutral charge and stops moving at that point aka it reaches its isoelectric focusing point.
What is a 2D gel? What advantages does this procedure have over 1D methods?
2D gels is applying both SDS PAGE and IEF gel methods to protein samples.
The advantage over 1D methods is that 2D gels can separate about 2,000 different proteins from a single protein sample. It can also be used to study all the proteins (proteome) expressed by a cell.
METHOD:
1) IEF gel is conducted first in a cylindrical tube shaped gel.
2) The tube shaped gel is pushed out of the tube then laid horizontally across the top of an SDS PAGE gel
3) Proteins are then separated using SDS PAGE on the basis of their size
4) The net result is that instead of bands you end up w/proteins in various spots (chocolate chip pattern)
How does immunoblotting enhance protein separation methods?
It allows you to see the location of just the proteins which were detected by an antibody. It is hard to analyze the amount of a specific protein of interest in other procedure and w/immunoblotting it is easy to analyze the specific protein b/c of antibodies
Describe the immunoblotting method
1) proteins are separated using either SDS or IEF
2) the gel is pressed against a sheet of paper or other flat material that has been treated w/reagents that make it stickier to proteins. This is typically called a membrane
3) different techniques are then used to transfer the proteins out of the gel and onto the membrane support w/o changing their positions. Usually some kind of apparatus is used to place another electrical field across the gel, and transfers the proteins to the membrane rapidly and efficiently.
4) The membrane is then incubated in a solution that contains antibodies which detect proteins of interest and then usually a second, a number of other methods used to identify the antibodies which have been bound to the membrane.
What was the original method used to transfer proteins out of the gel and onto the membrane support in immunoblotting? Hint: this gave immunoblotting its name.
Proteins were transferred by placing absorbent material on the other side of the membrane and more or less sucking the proteins through the gel and onto the membrane. This is known as blotting
What is an epitope tag? Why would you want to use one, and what might be some of the problems you encounter in doing so?
An epitope tag is a protein made with recombinant DNA methods. The epitope tag has the functional domains of the protein you want to study and then the tages bound to one end.
You might want to use an epitope tag b/c antibodies alone have limitations (such as if you have a rare protein you might not be able to produce an antibody for it and sometimes individual antibodies are expensive to produce) epitope tages could solve this problem.
Tags can vary in charge and size. This can be an issue b/c highly charged or very large epitopes can be used to facilitate biochemical purification of a protein but might interfere w/ the tagged proteins normal activity in cells.
What are some of the methods that you could use to study the function of a protein?
Gene replacement
Gene knockout
Gene addition
How are transgenic plants and animals produced?
Transgenic plants are genetically modified and transgenic animals are also genetically modified by introducing DNA into embryonic stem cells and egg
What methods can be used to determine the structure of proteins?
X-ray crystallography NMR Spectroscopy (Nuclear Magnetic Resonance)
How can we identify how much protein is in a cell?
It is difficult to simply look at a cell and tell how much of a specific protein is present.
Strategy: separate proteins on basis of size (SDS PAGE)
Strategy: Identify based on inherent electrical charges (Isoelectric Focusing)
What does SDS stand for? What are its properties and what is used for?
Sodium dodecyl sulfate
Properties: a negatively charged detergent
Used to incubate proteins aka coat all of the proteins in a uniform layer of negative in SDS PAGE
What happens when proteins are incubated with SDS?
DS detergent coats all of the proteins in a uniform layer of negative charges which makes the overall charge of the protein negative and the magnitude of the negative charge is directly related to how big the protein is.
What is polyacrylamide and when is it used?
jelly like plastic and it is used to make up gel material in SDS PAGE
Explain how protein size moves in SDS PAGE
The speed of the negatively charged proteins migrating towards positively charged electrode depends on the number of SDS detergent molecules that are stuck to the protein, which in turn is related to the size of the protein
Therefore, small proteins move faster than larger ones.
In an SDS PAGE diagram what do columns represent?
The proteins found in individual proteins samples.
Why are the smallest size proteins found at the bottom of the gel in SDS PAGE?
b/c small proteins can filter through the matrix of the gel faster than larger ones
what properties of a protein allow us to examine/separate proteins using isoelectric focusing gel electrophoresis (IEF gel electrophoresis)?
Proteins have inherent electrical charges
Proteins can accept charged ions found in their environment
Proteins can migrate toward or away from an electrode if they are charged
Proteins will not move at all if they are uncharged
What can the pH of a molecule tell us?
It can give us a description of how much positively charged or negatively charged ions are present on this molecule
What is a protein’s isoelectric focusing point?
The protein has no net charge and therefore no longer migrates in the electrical field.
In IEF gel electrophoresis, when a protein’s charge is cancelled out or equal the exact charge of the protein, the protein will reach a neutral charge and stops moving.
If protein is + charge, it will move toward the negative electrode and be surrounded by - charge in solution (protein becomes increasingly negative charged) until its charge cancels. If the protein is - charged it moves toward the positive electrode and is surrounded by + charge, (protein becomes increasingly positive) and charge will cancel and protein will stop moving.
In 2D gel, why is IEF gel method applied before the SDS PAGE?
IEF is done first b/c that technique is looking at the proteins own inherent charges which would be destroyed by the use of SDS
What are antibodies?
Can detect a single protein of interest
