Term
| What differences in amino acid composition would you predict for myosin and actin on the basis of their different salt solubilities? In other words, which amino acids would favor the highly polar environment of a high salt aqueous environment and which would favor a non-polar environment? |
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Definition
| Polar amino acids (both charged and uncharged) will favor a high salt aqueous environment. Nonpolor ones will not. |
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Term
predict the pedigree of each fraction
HSS:
HSP:
LSP: |
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Definition
HSS: good purity, actin absent, myosin present
HSP: good purity, actin present, myosin absent
LSP: good purity but less than hsp, actin absent, myosin present (more than hsp) |
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Term
| chicken breast is first diluted with __ to give __ and __. next the __ is diluted with __ to give __ and __ |
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Definition
| hight salt buffer; hsp (actin); hss(myosin); hss; low salt buffer; lsp (myosin); lss |
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Term
| Do the overall charges (pI) for the two proteins explain their different salt solubilities? |
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Definition
| Probably not, they are pretty similar. |
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Term
| What other factors might influence the salt solubilities of the two proteins, given that their overall charges are similar? |
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Definition
| The polar and non-polar residues may have different distributions on the two proteins. The polar residues may be concentrated in regions that are very polar in the myosin protein, making it more soluble in high salt. |
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Term
- __ with __ is best for separation of myosin
- __ with __ is best to seperate actin |
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Definition
- high salt []; low atp []
- low salt []; low atp [] |
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Term
- __ is predominantly neutral throughout
- __ has a highly hydrophilic C terminus |
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Definition
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Term
Actinomyosin contractile cycle: atp role
- atp hydrolysis energizes __ to __
- __ induces conformational change in __, causes __
- release of __ allows __ to bind
- __ allows release of __ from __ and new cycle |
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Definition
- myosin; bind actin
- release of Pi; myosin; sliding actin filament (power stroke)
- adp; atp
- atp binding; release; myosin head; actin |
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Term
| You will perform the electrophoresis of your fractions in today’s exercise and then carry out the analysis phase of the experiment in next week’s lab. The protein compositions of the fractions will be analyzed through both __ and __. |
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Definition
| coomassie staining; immunostaining |
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Term
| __ is protein-nonspecific and will allow you to visualize the entire protein composition of each fraction. |
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Definition
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Term
| __ is similar to the immunostaining technique used in Lab1B, in that it uses an antibody to detect a specific protein within that complex mixture of proteins. |
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Definition
| immunoblotting (aka western blotting) |
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Term
| First prepare your samples for the electrophoresis. The samples contain __ loading buffer. Boiling the samples in __ will cause the proteins to denature. The __ (See structure on pg. 129 of your text) will coat the surface of the denatured proteins, giving them a uniformly negative charge that will migrate toward the positive pole when subjected to an electrical current. |
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Definition
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Term
| Using the protein molecular weight standards on the Coomassie-stained class-wide gel as a guide, did the sizes of the bands that appeared on the two sets of immunoblots agree with the expected sizes for actin and myosin? |
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Definition
| The Myosin II heavy chain has a molecular weight of 150 kDa. Actin has a molecular weight of 40 kDa. The molecular weights of the proteins detected on the Western blots are pretty close to what is expected for each. (The 150 kDa molecular weight marker in the Protein Standards (Lane 1) is the Myosin II heavy chain.) |
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Term
| Judging from the actin and myosin immunoblots, how effective were your salt precipitations at separating actin from myosin in the chicken skeletal muscle. |
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Definition
| Because of the low solubility of actin in high salt, actin will probably remain in the pellet fraction (HSP, Lane 3) in the high salt extraction step. Because of the greater solubility of myosin in high salt, at least some myosin should have been extracted into the HSS fraction (HSS, Lane 2). There may also be some residual myosin found in the HSP fraction. We also expect myosin to become more enriched in the pellet fraction from the diluted HSS fraction (LSP, Lane 4). Therefore, if Lanes 2 and 4 were loaded with equivalent amounts of total protein, the intensity of the myosin band should be stronger in Lane 4 than Lane 2. |
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Term
| If you had done an immunoblot of all three of your group’s fractions on a single gel, as was done for the class-wide Coomassie-stained gel, can you think of any technical problems with the loading and electrophoresis of your gel that might have made the effectiveness of your separation of actin and myosin appear worse than it actually was? |
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Definition
| Gel loading problems, where proteins loaded into one well flow over into the neighboring wells, could make it appear like the separations were worse than they actually were in some cases. You can usually see evidence of this happening in the molecular weight lanes where you know the size of the protein bands expected. |
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Term
| Judging from the Coomassie staining profiles of the HSS and LSP fractions on the class-wide gel, how effective was the dilution of the High Salt Supernatant (HSS) at achieving a further purification of myosin (bringing about a higher concentration of myosin in the LSP fraction than in the original HSS fraction). |
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Definition
| A few differences between the protein profiles of the HSP and HSE fractions can be seen, but the Coomassie staining shows that salt extractions alone are not sufficient to purify a protein. They are only a beginning step. |
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Term
| Based on what you have learned in lecture about the actinomyosin contractile cycle in muscle contraction, what effect would adding ATP to the High Salt Buffer have on the extraction of myosin from the skeletal muscle? Would it be better or worse and explain why? |
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Definition
| ATP must be bound to the myosin head in order for the heads to dissociate from the actin filaments. Therefore, the addition of ATP should allow the removal of more myosin from actin filaments in the skeletal muscle. |
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Term
| Based on what you learned in lecture about ATP in both the actinomyosin contractile cycle and actin polymer assembly, speculate on how a high concentration of ATP might affect the extraction of actin from skeletal muscle. What effect would a low concentration of ATP have? |
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Definition
| ATP must be present for myosin heads to detach from the actin filaments, allowing dissociation of actin and myosin filaments. But high ATP concentrations also favor actin filament assembly into insoluble polymers. A low ATP concentration might both allow myosin detachment from the actin filaments and favor disassembly of actin filaments into soluble monomers. |
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Term
| You probably found that our protocol was fairly effective at extracting myosin from the skeletal muscle, but less effective at extracting actin. Based on what you have learned about the salt solubility of actin, relative to myosin, and the effects of ATP on actin discussed in question #6, devise a buffer composition (salt and ATP concentrations) that might be most effective in extracting actin from skeletal muscle. |
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Definition
| A low salt buffer should be used to optimize actin solubility. ATP concentrations must also be kept low in order to allow myosin detachment from the actin filaments and favor disassembly of actin filaments into soluble monomers. |
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Term
| Are the restriction fragment patterns of the three HOAP clones as expected? |
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Definition
| Yes The data provide clear evidence that a deletion occurred in Mutant 1. The point mutation in Mutant 2 cannot be visualized as a change in restriction fragment pattern. |
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Term
| How does the restriction digestion pattern of Drosophila genomic DNA compare to those of the three cloned HOAP genes? |
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Definition
| A smear of DNA fragments is observed in the digest of genomic DNA, making it impossible to determine which DNA fragment contains the HOAP gene. |
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Term
| If you wanted to analyze the restriction digestion pattern of the HOAP gene from its position in the Drosophila genome, what method would allow you to specifically label the DNA fragments from the HOAP gene only? (Hint: Recall how you were able to specifically label actin and myosin on your SDS PAGE gels in the previous lab.) |
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Definition
| You would have to use a labeled copy of the HOAP gene as a probe in a Southern blot to specifically label the HOAP gene. This is similar to using an antibody that recognizes the HOAP protein to specifically label the HOAP protein among the smear of proteins from a total protein extract (or to label actin or myosin in the extracts from skeletal muscle.) |
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Term
| Positioning the HOAP gene downstream of a bacterial promoter sequence in the plasmids allowed the proteins to be expressed in bacteria. How would you expect the mutations in Mutants 1 and 2 to affect the size and abundance of HOAP protein expressed from each of these clones? |
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Definition
These mutations would probably not affect the abundance of the protein, unless they affect their transcription, translation, or stability.
The mutation in Mutant 1 should produce a smaller HOAP protein. The mutation in Mutant 2 would probably not affect the size of the protein, unless it affects translation or stability of the protein. |
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Term
| Are the molecular weights of the two mutant HOAP proteins what you expected? How do you explain any discrepancy? |
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Definition
| The molecular weight of Mutant 1 protein is about one third the size of the wild type protein, and therefore, close to what is expected. The Mutant 2 protein was expected to be the same size as the wild type protein. This suggests that the point mutation in Mutant 2 is a nonsense mutation, which causes premature termination of translation to produce a truncated smaller protein. |
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Term
| How does the protein profile of the Drosophila Protein Extract compare to the purified bacterially expressed protein? |
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Definition
| It is much more complex. (Similar to the more complex Coomassie staining patterns of the skeletal muscle fractions, in comparison to the single protein bands observed on the actin and myosin Western blots.) |
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Term
| If you wanted to analyze the size and abundance of the HOAP protein in an extract from a Drosophila animal that you think may be mutant for the HOAP gene, what method could you use to target your analysis specifically to the HOAP protein in that extract? |
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Definition
| We would need to use anti-HOAP antibody in a Western blot |
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