2 experimental questions

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temmie

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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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Not an appropriate forum for getting your homework help.
 
Flopotomist said:
Not an appropriate forum for getting your homework help.

i just need some verification and i thought i could stimulate some brain storming
 
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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.
 
desiredusername said:
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.

Thanks, i figured they can go old school and use proteomics. Thanks for the good wishes, good luck to you too
 
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....
 
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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.
 
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
 
temmie said:
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.

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.
 
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
 
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.
 
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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