4 replies to this topic

[Taminem2012]

Hi,
  In many protocols involving some form of treatment on cells, for example immunostaining protocols, I run into many 'recommended' solutions that the protocols suggest me to use on the cells for certain steps such as a pre-extraction step for immunostaining MDCK's tight junctions.

Buffer:
* Pre-extract with 0.2% Triton X-100 in 100mM KCL, 3mM MgCl2, 1 mM CaCl2, 200mM sucrose, and 10mM Hepes (pH 7.1) for 2 min on ice, making sure that buffer is gently applied to cells.

I know this step is definitely crucial but for curiousity's sakes as well as for me to simply learn more about how certain solutions with certain ions and their concentrations affect - structure + physiology, are there any certain websites or books where I can learn more about how these solutions affect cells?

I am very weak in cell biology, and any recommendations that may help me learn more about cells is greatly appreciated.

Thanks for the time!

Edited by Taminem2012, 04 January 2012 - 10:47 AM.

[bob1]

I think you will find that it isn't so much about how the ions affect the cells, it will be more about how the ions affect the proteins they interact with.  In the case you described above, the triton (detergent) acts as a surfactant and helps puncture the membranes so that the rest of the solution can act more effectively as a hypo-osmotic solution, swelling the cell, and making the tight junctions more visible. The K, Mg and Ca are likely to be co-factors in the tight junction formation and/or permeability ( as a quick google search just confirmed).

[Taminem2012]

Hi bob1,
   You never cease to amaze me on how much you know! I'm having problems searching for how K, Mg, and Ca act as co-factors in tight junction formation - I'm a bad google searcher . If you don't mind, can I get some links to what you have found. I really appreciate the help, man!

[Taminem2012]

Also, as an experienced researcher as yourself, what are some resources that you regularly consult to to learn more about certain specifics that are normally difficult to find?

[bob1]

All I did was a google search for "tight junction potassium"  (without the quote marks and swapping calcium or magnesium in the place of potassium) and looked at some of the results that came up.  It helps to know a little bit of physiology with these sorts of things too - the K is used as part of an Na/K ATPase exchange pump.  Ignore the results that are from random websites - trust the ones that are from prominent journals and/or the NCBI/pubmed.

Resources that I use frequently are dependent on what I happen to be doing at the time, but good standbys are: Molecular Cloning: a laboratory manual (Sambrook et al.), Roche Lab FAQs (available free online, not all accurate though), Cell culture: a laboratory manual (Freshney), google ( learn to use the advanced functions like *, -, "your text here" etc), Hubmed (alternate pubmed interface, bit more user friendly and good for abstracts and exporting citations etc.), NCBI (of course - you can't escape it, and it has heaps of really good features for mol bio).

Other useful ones are: Merck index, Nucleic Acids Research list of electronic databases, NEB website, a healthy dose of skepticism, and a good general grounding in all the basic sciences and mathematics.