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lysis buffer not lysing adherent cells?


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8 replies to this topic

#1 philman

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Posted 08 February 2012 - 03:38 AM

Hi,

ok so I am new to western blotting (I know, I am a biochemist 2 years into a 4 year phd and never done a western blot before, half the rest of the lab cannot believe it either...) but I seem to have encountered a problem before I even start!

So I have my confluent cells in a 6 well plate, apply my conditions and timepoints, remove the media, apply 1ml ice cold PBS, remove, and apply 200ul lysis buffer. According to my fellow scientists showing me the technique, the cells should lyse quite fast and you should be able to see this under the microscope. However I look under the microscope and can still see my cells sitting there, fairly happily, still stuck to the bottom of the plate, still 10min after I have applied the lysis buffer.

In the end I used cell scrapers to scrape the cells off the bottom of the plate, and put the whole mixture, buffer, cell debris and all into eppendorfs and into the -80. But have no idea if this mixture is useless or what.


My main theory is that I am using adherent cells (HeLa, HMEC-1 (an endothelial cell line), and HUVEC (endothelial primary cells), whereas most of my labmates are pure immunologists and so are used to lysing non-adherent leucocytes.

Are adherent cells harder to lyse than non-adherent? or do they just look different when they do? or do I just need to use a different lysis buffer? people here doing monocytes or other leucocytes say that you can see the cells lysing under the microscope quite often and that it should happen fairly fast, are adherent cells just needing a slightly different technique?



Of course the first question any of you is going to ask me is what is the lysis buffer so here is the recipe I was given:

-50mM NaCl
-50mM Hepes
-1% Triton X
-1 tablet phosSTOP (phosphatase inhibitor)
-1 tablet protease inhibitors
-10ml H2O

(I am not sure of the brand of the tablets, this is my first time with westerns so just borrowing reagents from friend to get me off the ground for now)

#2 almost a doctor

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Posted 08 February 2012 - 06:28 AM

Comparing your buffer to what I usually use, I think the NaCl might be a bit low (my buffers are 150mM). Also, maybe you could add EDTA or EGTA (the buffer I currently use is 1mM of each of them), I think that might help detaching the adherent cells from the plastic.

Finally, I have a question. Are you sure 200µl of buffer are enough to cover the cells in a 6-well plate well? I usually grow my cells in 24 well plates, and use 100µl of buffer to lyse, so not sure if 200µl for a much bigger well is enough or not.

Just my 2 cents here.

#3 bithorax

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Posted 08 February 2012 - 06:55 AM

we routinely do lysis like this:
trypsinize cells, centrifuge, wash with PBS, centrufuge, resuspend in lysis buffer (10 min on ice).
it works good, especially if you want to use less lysis buffer

with your buffer I think you are lacking sodiud deoxycholate (0,5%), SDS wouldn't make any harm (0,1 %)
and as almost a doctor said more NaCl

good luck!

Edited by bithorax, 08 February 2012 - 06:55 AM.


#4 philman

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Posted 08 February 2012 - 07:23 AM

@biothorax. Surely the trypsin and centrifugation would affect sensitive cell signalling pathways? I am planning on looking at ERK later on which is produced in response to stress (such as centrifugation etc) and also as my main protein of interest is the cell surface protein E-selectin, trypsin will almost certainly affect its distribution and epitopes.

@almost a doctor. Ok I will try with more NaCl in my next try, and you do make a point about the volume. I usually culture cells in 6 well plates with around 2ml of media, so maybe I should try and go up to around 500ul of lysis buffer or maybe 1ml in order to completely cover them. But won't increasing this volume decrease the concentration of protein I get later on, affecting my ability to see them on a western blot?

any other responses welcome too!

Edited by philman, 08 February 2012 - 07:23 AM.


#5 almost a doctor

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Posted 08 February 2012 - 07:33 AM

Yes, increasing the lysis buffer volume will definitely decrease the concentration of your protein. Ideally you want to use the minimum possible, is always easier to dilute if you need to than trying to concentrate your protein.

How many cells / well are you growing? Is there any way you could grow them in smaller wells. Some commercial buffers recommend specific volumes depending on plate surface, or cell number, so you might be able to play with that (for example here: http://www.cellsigna...ducts/9803.html)

You should also consider what's the maximum volume you can load on your SDS-PAGE system, and how much total protein you want to load, so that you can have a better idea of how diluted can you allow your protein to be.

Hope this helps. Good luck!

#6 bithorax

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Posted 08 February 2012 - 07:38 AM

@ philman. you are right, trysinizing in your case is not the best idea.. good luck!

#7 scolix

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Posted 08 February 2012 - 07:55 AM

triton is a mild detergent and will help extract cytoplasmic proteins by permeabilizing cells. if cells are overgrown, it might be difficult to detach them by simply adding triton buffer.
if you really want the whole cell, use sds in lysis buffer.

#8 philman

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Posted 08 February 2012 - 07:55 AM

I havent counted the cellsto be honest, I just usually grow till they are confluent then use them, I will grow up a load more cells this week then and just try and lyse them under various conditions to see what happens. I could also try counting them too and see what they reccomend for that.

#9 philman

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Posted 08 February 2012 - 07:57 AM

and thanks scolix (posted about 3 seconds before I pressed enter onthe last post) I will try making a buffer with sds too then, or try and time my cells better.




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