Find content
Not Telling
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
#114124 Housekeeping protein issues
Posted
on 03 July 2011 - 08:01 AM
#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?
#79213 Trypsin/EDTA inactivation
Posted
on 13 July 2010 - 12:57 AM
laurequillo, on Jul 12 2010, 07:28 AM, said:
So it looks like I was wrong. Good to know!
I think you are not wrong since albumine and other bulk proteins in serum are targeted by trypsin and compete with cell surface proteins of cells
#77932 how can i bring cells onto a slide?
Posted
on 02 July 2010 - 11:13 AM
moljul, on Jul 1 2010, 11:22 AM, said:
how can i bring cells onto a slide without cytocentrifugation?
thanks for recommendation
thanks for recommendation
as suggested, seeding on cover glas is a good idea; round coverglas is to prefer for round wells of cell culture plates; if cells do not grow on glas, try coating with collagen, fibronectin, matrigel etc
#77936 wrongly used HRP-conjugated antibody in IP
Posted
on 02 July 2010 - 11:22 AM
[quote name='gyma' date='Jun 28 2010, 10:03 AM' post='77297']
I did IP and got no band but heavy and light chains, and samples include a group of positive control. does that mean the antibody didnt work?
I might have wrongly used HRP-conjugated antibody instead of non-conjugated one. But if so, I think protein G would not bind this antibody anymore and there should be no band at all. Am I right?
Thanks.
[/quote
Full binding of protein G may be hindered by HRP for steric reason; additionally, unspecific binding of proteins to HRP may increase unspecific precipitations
I did IP and got no band but heavy and light chains, and samples include a group of positive control. does that mean the antibody didnt work?
I might have wrongly used HRP-conjugated antibody instead of non-conjugated one. But if so, I think protein G would not bind this antibody anymore and there should be no band at all. Am I right?
Thanks.
[/quote
Full binding of protein G may be hindered by HRP for steric reason; additionally, unspecific binding of proteins to HRP may increase unspecific precipitations
#77775 Font for Power Point presentation
Posted
on 01 July 2010 - 09:19 AM
Which font for headlines and/or other text is state of the art for ppt?
I prefer Microsoft sans sarif but I do not know if itīs still fresh...
I prefer Microsoft sans sarif but I do not know if itīs still fresh...
#77588 Who are the most important scientists in life science currently?
Posted
on 30 June 2010 - 02:30 AM
in each life science discipline there are, of course, some leaders but who is most important beyond her/his faculty?
"Importance" can be discussed in various directions: importance in science, or importance in making a topic popular or important to explain us science or etc
any contribution is appreciated
to drop a name, my favourite is Hiroaki Kitano
"Importance" can be discussed in various directions: importance in science, or importance in making a topic popular or important to explain us science or etc
any contribution is appreciated
to drop a name, my favourite is Hiroaki Kitano
#76806 Sample loading buffer turning yellow.
Posted
on 24 June 2010 - 02:29 AM
Aravin, on Jun 23 2010, 10:48 PM, said:
I added sample loading buffer (Bromophenol blue) to my sample and after boiling for 5 min, it turned yellow. Does anyone know the explanation behind this?
Thanks.
Thanks.
Actually, BPB is a pH indicator turning yellow at acidic pH; so, your sample contains some protons which were not buffered by the sample buffer. Was it blue before boiling? Why so long boiling?
#76396 clumping of cells
Posted
on 21 June 2010 - 12:29 PM
sagagirish, on Jun 21 2010, 05:42 PM, said:
Hi....i have been growing HeLa cells ....they look fine...but when i trypsinize them,,,most of them detach to form single cells..however some huge clumps of cells are formed ...and this causes loss of huge amount of cells..how is it that there are no clumps initially and later clumps are formed?
Is this bad ?
Thanks
Is this bad ?
Thanks
do you wash cells with serum-supplemented cell medium to inactivate trypsin? normally, albumine reduces clumping...
#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!
#112929 brain tissue preparation for immunoblot
Posted
on 19 June 2011 - 02:48 AM
how do you dissect brain tissue for immunoblot analyzis, especially if you like to analyze phsophoproteins? I do not mean LASER dissection but with scalpel and binocular... any suggestion is welcome!
#112177 Brain dissection for immunoblot
Posted
on 09 June 2011 - 02:20 AM
how do you dissect brain tissue for immunoblot analyzis, especially if you like to analyze phsophoproteins? I do not mean LASER dissection but with scalpel and binocular... any suggestion is welcome!
#110826 western blot bands wider at the middle than the top
Posted
on 25 May 2011 - 04:03 AM
Mw and Rf do not behave linear but exponential in non-gradient gels, so your observation reflects this
swatcats, on 24 May 2011 - 02:30 PM, said:
Hi
Maybe this is very basic...but I can't figure this right now. When I use a 7.5% precast gel, the marker bands run quite smooth but when I do the same thing on the 10% gel the marker bands are smoother at the top but seem to get wider at the bottom. I have now noticed this for a couple of times only with the 10% gels. Both are from BioRad and are brand new. Could anything be there with the buffer not mixing well? Or the voltage? I use the tris-glycine-SDS buffer (10x diluted to 1x as with the other gels) and the voltage is always between 90-110.
Thanks
Maybe this is very basic...but I can't figure this right now. When I use a 7.5% precast gel, the marker bands run quite smooth but when I do the same thing on the 10% gel the marker bands are smoother at the top but seem to get wider at the bottom. I have now noticed this for a couple of times only with the 10% gels. Both are from BioRad and are brand new. Could anything be there with the buffer not mixing well? Or the voltage? I use the tris-glycine-SDS buffer (10x diluted to 1x as with the other gels) and the voltage is always between 90-110.
Thanks
