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yimao's Content

There have been 9 items by yimao (Search limited from 23-January 19)


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#112950 how many samples should i prepare?

Posted by yimao on 19 June 2011 - 09:28 AM in Protein and Proteomics

oh, sorry about my bad expression.

i am trying to compare the protein expression differences made by culture cell in difference drug treat conditions.

do you mean i need to run 3*3=9 gels for one cell culture condition or 1*3=3 gels?

thx~

your request misses the aim of your 2D study so it is difficult to answer


generally you should run at least n=3 gels of 3 different cell cultures




Hi, i am plan to do a 2-DE experiment on cells cultured in different conditions.

May I ask how many sample/s do i need to prepare for one condition (of cell culture)? Because some papers said that we will need to run 3 gels for "each sample", is that means i need to collect three samples from cell culture or only need to collect one cell sample, but run 3 gels by proteins extracted from this sample?

Thanks!




#112879 how many samples should i prepare?

Posted by yimao on 17 June 2011 - 09:20 PM in Protein and Proteomics

Hi, i am plan to do a 2-DE experiment on cells cultured in different conditions.

May I ask how many sample/s do i need to prepare for one condition (of cell culture)? Because some papers said that we will need to run 3 gels for "each sample", is that means i need to collect three samples from cell culture or only need to collect one cell sample, but run 3 gels by proteins extracted from this sample?

Thanks!



#112425 cell homogenization

Posted by yimao on 12 June 2011 - 08:26 PM in Cell Biology

hey, i was also looking for a grinder to grind my cell line pellet, and i found this online, is it fit your purpose?

do u know what is the difference between "Large clearance" and "Small clearence" means?

http://www.sigmaaldr...blePage=9578358


Hi Bob1, thank you so much for the suggestion. I actually had no idea of how that looked like so I googled it. It looked like this?

http://www.bio-world...able-mL-PK.html

Do you happen to know of how to use them? Like how many strokes to fully homogenize them? I have thought about using QIAshredder for homogenizing the cells, it's those patented microfuge tubes where you just add your samples on top and centrifuge them and you get homogenized samples on the bottom tube, but I think it is going to be really costly since I could only run one sample per tube ($84 for 50 tubes).

I also thought that sonication or freeze thawing would be a lot more time-efficient and economical, but I can see your point about freeze thawing reducing the enzyme activity. I am also worried that using the pestle for manual homogenization may not give as much as uniform treatment as homogenization or freeze thawing?

I think what I need to do is run a small preliminary trial and see the recovery of the enzyme activity with these methods. What do you think?




#111864 will this method break organelles?

Posted by yimao on 04 June 2011 - 07:52 PM in Cell Biology

hi, guys, i was doing lysis of cultured mamallian cell for 2DE studay, and i used Lysis Solution with the composition of:

8M urea, 4% w/v Triton X-100 (a mild detergent)
then votex for 5 min

my Q is:
will this method break organelles like: necleus, microbodies, mitochondrial and golgi apparatus?
or am i just get to the cytosol only?

thx~



#111761 what can 4% Triton X-100 or CHAPS do to cultured mammalian cell as lysis agent

Posted by yimao on 03 June 2011 - 07:47 AM in Protein and Proteomics

can they break the nucleus or any other organelles in the cell?

can they extract and dissolve membrane proteins in all the membranes?

thx~



#104359 Which wash buffer (to collect cell) is prefered for 2DE?

Posted by yimao on 21 March 2011 - 07:20 PM in Protein and Proteomics

tris+sucrose has lower ionic strength (presumably, since you didn't give any concentration information) so will have less effect on electrophoresis.

if we don't dry them, we store our gels in coomassie destaining solution (30%methanol, 7% acetic acid). the balance of methanol and acetic acid maintain the physical dimensions over the long term (as long as you occasionally replace the solution).


thx, man.

i use 0.6g Tris, 42.8g Sucrose dissolve in 500ml h2o, then adjust the ph to 7. (10mM Tris, 250mM Sucrose, pH 7.0)

could coomassie destaining solution also preserve the protein in gel as long too? kinda need to do some MS protein identification after that.



#104191 Which wash buffer (to collect cell) is prefered for 2DE?

Posted by yimao on 20 March 2011 - 12:05 AM in Protein and Proteomics

PBS or the one with Tris + Sucrose? thx~

btw: any good method to store the SDS gel after silver stain? i mean for a longer period of time, like months?



#103904 can i use trypsin to collect cell sample for 2d gel analysis?

Posted by yimao on 16 March 2011 - 07:26 PM in Protein and Proteomics

you can use trypsin as long as you wash the cells thoroughly before you lyse them.


u mean after collect the cell by trypsin, right?

3 time by Wash Buffer good enough? :)



#103784 can i use trypsin to collect cell sample for 2d gel analysis?

Posted by yimao on 16 March 2011 - 01:38 AM in Protein and Proteomics

can i use trypsin to collect cell line sample from plate for 2d gel proteomic analysis? would that influence much about the result of experiment?

or should i only use a "rubber police"?

thx~




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