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Without permissing first phosphorylation, second phosphorylation may occur to lower extent; only second-site phosphorylation may have different result in kinase properties than first+second-site phosphorylation;
For first site phosphorylation you may need, f.i. second messenger activation; if it is blocked or missing, second-site phosphorylations increase
are the phosphorylations which you are analyzing mainly cis- or trans-phosphorylations?
#129385 Sequential phosphorylation in vivo but not in vitro
Posted
on 17 February 2012 - 06:51 AM
#116032 How the heck does serum activate Akt?!?
Posted
on 26 July 2011 - 03:33 AM
how do you run controls? how do you check activation of Akt?
#114889 5M NaF won't dissolve!
Posted
on 12 July 2011 - 11:32 AM
high salt solution keeps for a while non-contaminated but better with NaN3 at RT
#114785 Dark western blot film
Posted
on 11 July 2011 - 09:20 AM
was it an anti-phospho antibody? milk contains a plenty of phospho-caseins...
#114784 protein precipitation
Posted
on 11 July 2011 - 09:15 AM
What I do not understand is why TCA is used for resuspension? For SDS-PAGE a Leammli buffer is to use
#114683 Dissolving Calcium Chloride in PBS: How to Prevent Precipitation?
Posted
on 09 July 2011 - 12:12 PM
in addition to bob1: add stock solution of CaCl2 stepwise to PBS under stiring
#114681 Repellent of glasses for PAGE
Posted
on 09 July 2011 - 12:08 PM
I think Sigmacoat is a silane diluted in organic phase; it works for glass but not alox plates (Hoefer); plates must be cleaned, then coated by rubbing a portion with cloth, shortly rinsed with distilled water and dried;
if Sigmacoat does not work in your case it seems to be too diluted for your purpose; you may repeat coating twice or more;
or buy pure silane (Sigma)and dilute with CCl4
if Sigmacoat does not work in your case it seems to be too diluted for your purpose; you may repeat coating twice or more;
or buy pure silane (Sigma)and dilute with CCl4
#114361 How to open *.bin?
Posted
on 06 July 2011 - 06:25 AM
I have got a file with bin extension but I donīt know the program to open. Any idea, please?
#114132 How to be good at the bench
Posted
on 03 July 2011 - 08:41 AM
being good at bench needs various inputs;
however, an important thing is:
when I started with lab work as a student assistant I tried to learn from EVERYONE in the lab; so, a close contact to your lab mates is essential i.m.p.o.v.
however, an important thing is:
when I started with lab work as a student assistant I tried to learn from EVERYONE in the lab; so, a close contact to your lab mates is essential i.m.p.o.v.
#114125 Detecting phosphoproteins
Posted
on 03 July 2011 - 08:14 AM
induced phosphorylations normally exhibit typical kinetics which is not a linear increase but Koshland kinetics; so it is extremely important to have the kinetics of phosphorylation under control
donīt you think that parallel use of two antibodies may affect each other? the polyclonal may compete with the monoclonal for the same epitope?
donīt you think that parallel use of two antibodies may affect each other? the polyclonal may compete with the monoclonal for the same epitope?
#114124 Housekeeping protein issues
Posted
on 03 July 2011 - 08:01 AM
ischamic and hypoxic situations are serious situations for cells and induce multiple processes; I think that it is possible to affect expression of so-called house-keeing genes; if you have the possibility check tubulin mRNA expression with PCR or take total protein amount as reference
#113676 Reference protein for membrane proteins in SDS-PAGE
Posted
on 28 June 2011 - 06:18 AM
often used reference proteins for cytosolic proteins are b-actin or a-tubulin; however, which reference protein to use for membrane fraction? receptor proteins are not suitable as they normally have a vivid turnover. what about membrane skeleton proteins (ankyrin, spectrin, ezrin protein family, other actin-binding proteins etc)? Or is also b-actin to use as it is linked to the plasma membrane?
#113670 Does LY means Lilly?
Posted
on 28 June 2011 - 06:07 AM
some pharmacologiacl substances are abbreviated as LY with a code number (f.i. LY 294002); does LY stand for Lilly?
#112932 Preparation of mammalian cell lysis
Posted
on 19 June 2011 - 03:04 AM
sansub, on 18 June 2011 - 07:03 PM, said:
What if I do not centrifuge the cells after adding the protease inhibitor and cell lysis buffer to them? Please help!
then you have full lysate with organelle debris, DNA and cytoskeletons; you may get problems with dissolving this kind of lysate with SDS sample buffer
#112930 how many samples should i prepare?
Posted
on 19 June 2011 - 02:55 AM
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
generally you should run at least n=3 gels of 3 different cell cultures
yimao, on 17 June 2011 - 09:20 PM, said:
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!
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!
