Term
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Definition
| The movement of charged molecules in an aqueous environment due to the effect of an applied electric field |
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Term
| Electrophoretic materials |
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Definition
| paper, starch, cellulose acetate, agarose, polyacrylamide |
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Term
| Draw electrophoresis apparatus |
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Definition
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Term
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Definition
| highly purified polysaccharide derived from agar. |
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Term
| By which property is agarose useful? |
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Definition
When heated, the polysaccharide molecules dissolve in aqueous solution and fully extend;
When cooled,the polysaccharide strands fold and twist up with each other, forming a meshwork |
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Term
| What are the concentrations by which agarose can be varied. How does concentration affect properties of gel. |
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Definition
| 0.5 to 3 %. The higher the concentration, the smaller the pores will be in the solidified gel. |
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Term
| What charge are RNA, DNA, and proteins |
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Definition
| RNA and DNA are always negative (because of phosphate groups). Proteins may either be positive, negative, or relatively neutral |
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Term
| What is used to visualize DNA/RNA. How does EtBr increase fluorescence |
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Definition
| Ethidium-Br. DNA increases fluorescence by 25 fold). By intercalation which allows it to stack between base pairs and lengthen and untwist the DNA helix |
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Term
| What mixes with acrylamide to make polyacrylamide and what is polyacrylamide gel good for |
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Definition
| TEMED and Ammonia Persulfate. Polyacrylamide is good for protein analysis but also nucleic acid analysis |
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Term
| What is name for poly acrylamide cross linker |
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Definition
| BIS(N,N’-methylene bisacrylamide): |
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Term
| Describe stacking polyacrylamide gel |
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Definition
| Has a resolving gel layer and a stacking gel layer. buffers used to prepare gel layers are of different ionic strengths and pH. stacking gel has a lower acrylamide concentration so its pore sizes are larger. |
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Term
| Draw diagram of stacking polyacrylamide gel. Why does stacking gel have large pores? |
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Definition
| Stacking gel causes proteins to be concentrated into a tight band during the first few minutes of electrophoresis. Proteins pass through stacking layer to get resolved in resolving layer. |
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Term
| Proteins in SDS are what charge? |
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Definition
| Negative proportional to size. Solves issue of charge inequality between proteins |
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Term
| 2 ways to visualize proteins |
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Definition
Coomassie Blue (detect 100 ng/band)
and Silver nitrate (detect 1-10 ng/band) (see reduced silver with silver nitrate) |
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Term
| What is SDS PAGE used for? |
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Definition
Determine protein purity
Determine molecular weight of proteins |
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Term
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Definition
| distance migrated by protein/distance migrated by marker |
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Term
| Relation ship between mobility and molecular weight |
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Definition
| log molecular weight = (slope)(mobility) + y-intercept |
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Term
| Draw Western blotting Diagram |
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Definition
| Refer to presentation4 slide 15 |
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Term
| Describe a western blot reaction |
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Definition
| after a SDS PAGE electrophoresis process has occurred. Proteins are transfered to a nitrocellulose membrane. This substrate is coated with a particular antibody which identifies a specific property of proteins (uses specific epitope (V part)). The target protein is targeted by an antitarget antibody this antibody is targeted by a second antibody. The second antibody is targeted by a horse-raddish enzyme This enzyme enables the visualization of the specific proteins |
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Term
| What dye can't be used with western blot and why? |
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Definition
| Coomassie blue, because the antibodies will be denatured by acidic conditions |
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Term
| What is the advantage of using 2D gel electrophoresis? |
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Definition
| 2D gel increases resolution and power: it enables proteins to not only by size separated but also by isoelectric point of protein. Must be run twice and in a tube. May also be used in MS for MW and composition determination. |
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Term
| What is a supershift and what is it used for? |
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Definition
| A supershift occurs when a specific antibody attaches to a DNA/protein complex. It is used to specifically identify a protein that attaches to a target DNA strand (probe). It causes the complex to become larger, which decreases its mobility, thus, it travels less than the rest |
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Term
| What are the three ways to purify proteins based on size |
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Definition
Dialysis - used to purify protein, by removing salt by a membrane (bag). The membrane has holes large enough to diffuse out salt and water, but not protein.
Centricon centrifugation - Uses a tube that acts a membrane contained in another tube. Centrifugation makes the salt and water migrate to the bottom of the large tube, while the small tube contains the purified protein
Gel Filtration Chromatography-Smaller molecules are captured by the beads, which acts like small mazes that the smaller molecules have to pas sthrough. Larger proteins do not go through beads, and elute out faster |
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Term
| Describe Purification based on specific binding. |
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Definition
Can be performed with different stationary phases:
DNA
Sugar
Specific substrate (ATP)
Protein partner
2. Also can use Tags to attach to sample to separate. Tags include:
His
GST
MBP |
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Term
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Definition
| Histidine side chains interact with nickel. This nickel will also bind to the stationary phase. To get this exact geometry there needs to be 4-6 His tags on protein. Thus, usually a 6-10 histidine tag is attached to the protein that we are trying to purify. Then imidazole is added and competes with protein for nickel. After this occurs protein elutes out. This process is usually repeated 2-3 times |
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Term
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Definition
| Purification based on polarity is the better method, but it requires already fairly pure sample in order to work, because polarity is a weak interaction. Is opposite of another method, because you actually add more salt, rather than less |
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Term
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Definition
Purification Based on Protein Charge
makes approx. 70% pure sample
We need sample that is 90% pure for enzyme kinetics, and over that for making crystals |
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Term
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Definition
| Isoelectric point – pI, pH at which net charge of proteins are charge. If pH is lower than pI net charge is positive if pH is higher net charge is negative |
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Term
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Definition
| Ionic precipitation by using ionic salts is easiest way to purify proteins |
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