8 replies to this topic

[SF_HK]

Hi,

I'm new to western and would appreciate all the tricks to save time.  The first time I did Western, I did cell lysis using

1) Add RIPA + protease inhibitor to cell pellet, incubate on ice for 15min, centrige at 13000rmp for 15min, collect supernatant
2) measure protein concentration
3) Add SDS loading dye and boiling for 10min at 100deg.

This is quite along and tedious process. Someone suggested direct protein lysis by simply
1) adding the SDS loading dye to the cell pellet, mixing
2) boiling at 100deg for 10min

But by doin this I get a sticky solution, is that normal?  Can I doa direct lysis using  only RIPA?

[madrius1]

The first protocol is already a shortcut to the normal lysis method I use..

I don't see anything you could do faster. And measuring protein concentration seems very important, as not doing it could result in false results, or having to do the Western again with adjusted volumes..

2x SDS lysis becomes viscous because of the DNA.

Best wishes

[Dr Teeth]

The first protocol is the best route.  If this seems too cumbersome for you, I don't recommend ever doing ChIPs!

[mastermi]

Depends on what you need.

I usually simply cook my cell pellets in sds loading dye, where I use 10 µl of loading dye per od=0.1
But then you have to check if your protein amounts are comparable by making two gels (one for staining, one for blotting) or by staining your gel with ponceau s.
This always worked fine for me...

[Penguin]

I sometimes do the shortcut of adding SDS-loading buffer straight onto my cells and have found the stickiness is reduced if you boil for 15-20 mins before SDS-PAGE

[sssss]

Penguin, on Apr 9 2009, 03:54 AM, said:

I sometimes do the shortcut of adding SDS-loading buffer straight onto my cells and have found the stickiness is reduced if you boil for 15-20 mins before SDS-PAGE

  by boiling the cells  its difficult to load in the gel.............
give high spin for 5-6 minutes.......its easy to pipette and load

[laurequillo]

I use both protocols (and normally I dont check amount of protein, as I use the same amount of cells and I am not working with cell death or apoptosis or something that kill my cells).
If I want to see some specific protein modification (as sumoylation) I use laemli directly on the samples, but to avoid the viscosity that is how I do it:

Recolect the cells
Add laemli buffer 1x 120ul
Boil for 2 minutes
Sonicate 2x15sec
Boil 5 minutes

I have no problem with this easy protocol

[RNA]

How about DNase to chew up DNA?  I have some DNase from a transcription kit which seems to work quickly.
I have ~1 mL samples.

How about centrifuging the samples to remove aggregates and debris?  

Edited by RNA, 19 January 2011 - 09:15 PM.

[RNA]

Thanks Batman, your protocol worked like a charm!  Solution was not viscous at all.

laurequillo, on 25 September 2009 - 04:34 AM, said:

I use both protocols (and normally I dont check amount of protein, as I use the same amount of cells and I am not working with cell death or apoptosis or something that kill my cells).
If I want to see some specific protein modification (as sumoylation) I use laemli directly on the samples, but to avoid the viscosity that is how I do it:

Recolect the cells
Add laemli buffer 1x 120ul
Boil for 2 minutes
Sonicate 2x15sec
Boil 5 minutes

I have no problem with this easy protocol