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2 experimental questions

Discussion in 'Pre-Medical - MD' started by temmie, Dec 1, 2005.

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  1. temmie

    temmie Senior Member
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    1) Can you think of an experiment to carry out that would test to see if a protein is a surface protein.
    2) Describe an experimental approach that could be used to obtain a protein sequence.
     
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  3. Flopotomist

    Flopotomist I love the Chicago USPS
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    Not an appropriate forum for getting your homework help.
     
  4. temmie

    temmie Senior Member
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    i just need some verification and i thought i could stimulate some brain storming
     
  5. jebus

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    um, what kind of imaging have you learned in class? (cough*ICC*cough)
    and do they teach edman degredation anymore? to be fair, it would be easier and probably cheaper to just ask sigma to find out the sequence. I wish i were kidding. working in labs is crazy like that. tell it to your professor, i want to find out if they teach you practical answers.
    this is crazy, these questions were on my biology 297 test in 1999. wow, i feel old.
    and flop's right, get someone else to do your homework. and good luck at your USC interview.
     
  6. temmie

    temmie Senior Member
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    Thanks, i figured they can go old school and use proteomics. Thanks for the good wishes, good luck to you too
     
  7. jebus

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    proteomics is decidedly new sk00l. and that's like the most expensive time consuming way and i'm not sure if it would tell you if you have a surface protein. all you want to do is put a tag on the protein with an antibody and see if the cell surface lights up under a fluorescent microscope.... if the cell periphery lights up the protein is on the surface....i'm not sure how proteomics would work at all. you wouldn't have a cell: you would have cellular gemisch on a slide....
     
  8. temmie

    temmie Senior Member
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    sdrrtdtfkufuyfyifvk
     
  9. jebus

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    hoping won't make it so. just write this down and then learn what the hell it means: 2-D sds-page and then some mass-spec, or De novo repeat detection sequencing, or peptide mass fingerprinting on the excised gel fragment.
    it's like college all over again. i'm the one teaching people the course and you'll probably do better on the test than me, too.
     
  10. toxin

    toxin Senior Member
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    surface proteins are hydrophilic while the ones on the inside are hydrophobic...(generalization)...utilizing this once you determine the sequence of the protein , then u determine the 3d structure which is at best very poor but there are general rules that increase the accuracy to about 70%...(such as glycine will induce a kink ...beta sheets have a repeating sequences etc)....so on and so on ....im sure you get the picture
     
  11. dr.z

    Physician 10+ Year Member

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    1. Over express the protein with tags (ie: HA, FLAG, Myc and so on) by transfection. Do immunostaining to see where the protein localized.

    2. You can do proteolytic digests with proteases and get the digestion pattern. They cleave proteins at specific sequences. You can also use mass-spec to determin the sequence of protein which is now very common method.
     
  12. chicklett1

    chicklett1 Senior Member
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    1. Since surface proteins are hydrophillic, you could use deuterium exchange (wash the sample in heavy water, and the protein will exchange its hydrogens for deuterium) and then you have 'tags' for what areas are open to exchange with the solvent.
    2. I'd go with edman degredation
     
  13. potato51

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    You can do immunostaining for surface proteins. Not sure exactly how, but I think if you can visualize the protein without permeabilizing the membrane, it's probably surface.
     
  14. threemusketeers

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    wouldnt the simplest way be to lyse the cells, centrifuge, and then run a gel? if your protein is in the soluble fraction, it is not membrane protein, if it is in the inclusion bodies, it is a membrane protein.
     
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