Tools of Protein Biochemistry Flashcards
What are two major types of electrophoresis
- ) Native-PAGE (non-denaturing)
2. ) SDS-PAGE (denaturing)
Separates molecules according to their charge, shape, and size
Electrophoresis
How many proteins are expressed in our bodies?
20,000
Buffer pH and intrinsic chemical properties of macromolecules (DNA, RNA, and proteins) determine their
Net charge
Electrophoresis requires a gel for
Support and Molecular sieving
What does PAGE mean in SDS-PAGE?
Polyacrylimide gel electrophoresis
Electrophoresis where the protein retains it’s activity throughout the process
-Non-denaturing
Native-PAGE
Polypeptides retain their higher-order structure and often retain enzymatic activity and interaction with other polypeptides in
Native PAGE
In native PAGE, the migration of proteins depends on
Size, shape, and native charge
Single amino acid substitution of Glutamate 6 with valine
-causes conformational change that affects hemoglobins ability to bind oxygen
Sickle Cell Disease
Hemoglobin A is a tetramer composed of what 4 subunits?
2α and 2β
What significance does the fact that a glutimate (negative charge) is substituted for a valine (neutral charge) in people with sickle cell?
HbA and Hb-S can be separated at physiological pH because HbA is negatively charged
Which Hb migrates faster during electrophoresis at pH 7?
HbA (it is negatively charged)
Which Hb migrates faster during electrophoresis at pH 4?
HbA and Hb-S have the same charge and thus can not be separated
Electrophoresis method to separate proteins baed on molecular weight
SDS-Page
Prior to SDS-PAGE, proteins are treated with which two things?
- ) Reducing agent to break disulfide bonds (β -mercapthanol)
- ) SDS
An ionic detergent that denatures secondary and non-disulfide-linked tertiary structures
-Binds to protein backbone and confers a negative charge proportional to mass
Sodium dodecylsulfate (SDS)
What fragments moves faster in SDS-PAGE?
Small fragments
The movement in SDS-PAGE is not linear, but rather
Logarithmic
Cyclists, distance runners and crosscountry skiers have been utilizing recombinant human erythropoietin (RhEPO) to improve their endurance performance. How can we detect Erythropoietin?
SDS-PAGE
Induces hemoglobin production which allows more oxygen to be carried in the blood and improves endurance
Erythropoietin
Erythropoietin was created for people with anemia or people undergoing chemo to help increase
Hemoglobin production
Erythropoietin (EPO) is a hormone produced by the kidney that promotes the formation of
Red blood cells by bone marrow
Blood does not have many circulating proteins in it, but in the cell their are many. How can we isolate a specific protein of interest?
Western Blot
In all types of blotting,we make our molecules negatively charged so that we are separating exclusively by
Size
How do we generate antibodies that bind our target protein in a western blot?
Inject a rabbit with the antigen (target protein). Take serum from the rabbit. The supernatant will contain the desired antibodies
Portion of a molecule to which an antibody binds
Epitope
The antigen binding site of an antibody
Paratope
Epitopes can be composed of
Sugars, lipids, or amino acids
Antibody with a bound enzyme that can catalyze
conversion of a colorless molecule to a colored one
Indicator
What are two common indicators?
Alkaline phosphatase and Horseradish peroxidase
Summarize a western blot
The antibodies specific to the protein of interest are generated by injecting a rabbit with the antigen (protein of interest) and taking blood serum from the rabbit. Next, gel electrophoresis is performed on the sample containing our target protein. Upon completion, the wells are transferred from the gel to a nitrocellulose sheet. The antibodies we generated are added to the sheet, at which point they bind the target protein. All other antibodies are washed way and tagged antibody is added (indicator), which allows us to isolate the desired protein.
Combine electrophoresis and immunological techniques
Western Blots
First thing you do when screening for a disease. A faster, more sensitive method than western blotting to detect antibodies or antigens in a patient
Enzyme-Linked Immunoabsorbant Assay (ELISA)
Antibodies developed by the human body against AIDS can be detected by
ELISA
Troponin, released after a myocardial infarction, can be detected by
ELISA
Which is more specific, Western Blotting, or ELISA?
Western Blotting
Explain how ELISA works
You have a well, to which you can link an antigen or an antibody, depending on which one you are testing for. Next, add an enzyme antibody, which will bind to the antigen and allow for detection.
Tagged antibodies bind directly to the antigen in the well and allow for detection in?
Direct ELISA
A primary antibody binds directly to the antigen in the well. Then, tagged secondary antibodies bind to the primary antibody and allow for detection in?
Indirect ELISA
How is the antigen actually detected in a western blot or ELISA?
A substrate is added that binds the enzyme attached to the antibody and produces either a fluorescent or colored product
ELISA is more sensitive than a western blot because?
The well is hooked up to a spectrophotometric detector. Where as in a western, we have to observe the color change with our eyes (requires a larger sample)
What is the gold standard for HIV testing?
Perform an ELISA, if it’ negative than the patient is negative. If it is positive, perform a Western blot for confirmation. The blot results will provide the official diagnosis
The time between HIV infection and when antibodies to HIV are produced
Window Period (4-6 weeks)
What indicates a positive HIV Western Blot?
You need one band to be present in the region of gp 160 or gp 120, and one band present in the region of p31 or p24
When bands are present but the pattern does not meet the criteria for positivity, the result is
Indeterminate
What are some causes of an indeterminate HIV western blot?
Recent infection, advanced HIV, certain strands of HIV, etc.
What is the procedure following an indeterminte result?
Retest in > 6 weeks
What does the Western blot HIV test actually measure?
The antibody to HIV
What are the antibodies to HIV bound to?
Antigen to HIV
Inadequate blood supply to an organ or part of the body
-Cause of myocardial infarction
Ischemia
Serve as useful diagnostic markers of acute ischemia
Troponin, Myoglobin, and Creatine Kinase
Cardiac cells rupture and release proteins into the blood. As a consequence, the levels of Troponin T increase, which indicates
Myocardial infarction
The contractile regulating protein of striated muscle
Troponin
The cardiac isoforms of troponin T and I increase in serum after
Acute Myocardial Infarction
Troponin is highly specific for myocardial infarction because it is not elevated in any other trauma, heavy exercise, or renal failure. Also, it remains in the blood for up to
Four days following the event
What is the difference between HIV ELISA and Troponin ELISA?
Troponin ELISA uses an immobilized antibody in the well. Where as HIV used an immobilized antigen
Describe how co-immunoprecipitation works
We bind beads to an antibody specific to our target protein and add it to our sample. The antibody binds the target protein.The increased mass from the beads causes the complex to precipitate out. If our target protein is complexed with another protein, that protein will also precipitate out. We can separate the two by SDS page and then perform a western blot
Allows the proliferation of damaged cells, which can lead to tumor development
Loss of p53
p53 tumore suppressor is frequently disabled by
Mutations
Wild type in 50% of human cancer
p53 gene
In response to cellular stress, promotes p53 ubiquination and degredation by the proteasome
-An E3 ubiquitin ligase
MDM2 (HDM2)
Targets p53 for degredation to disable the p53
Overexpression of MDM2 in human cancer
How did scientists show that MDM2 and p53 interact?
Immunoprecipitation of MDM2 which showed coprecipitation of p53
Describe immunoaffinity Chromatography
We add antibodies specific to our target protein to a column. We then add a cell extract to the column. As the cell extract moves through the column, the target protein will bind the antibodies and everything else will be eluted. Then we prepare a solution to elute our target protein, which we can then load to the gel and quantify
Purifying proteins from cell extracts
Affinity Purification
Can immunoaffinity chromatography be used for interaction proteins similar to how co-immunoprecipitation worked?
Yes
Uses primary antibodies to target and label specific proteins in tissue and then secondary antibodies with a conjugated enzyme to bind the primary antibodies
-Key is that it is in the tissue
Immunohistochemistry
A breast cancer that tests positive for a protein called human epidermal growth factor receptor 2 (HER2), which promotes the growth of cancer cells
HER2-positive breast cancer
In about 1 out of every 5 breast cancers, there is a gene mutation that results in the roduction of excess
HER2
Tend to be more aggresive than other types of breast cancer
HER2-positive breast cancers
Added to molecules that an investigator wants to visualize when we can’t develop an antibody for our protein of interest
Epitope tag
How does epitope tagging work?
We make and add a tag to the DNA sequence of the desired molecule, which results in the expressed protein being made with the tag in its amino acd sequence. We can then generate an antibody specific to the tag and detect with a blot
