Protein Extraction SDS-Buffer Pellet not Dissolving and Laemlli Bufffer Green? - (Sep/03/2011 )

I've just completed a protein extraction from plant suspension cells and ressupended the pellets in SDS running (Laemlli) buffer but neither pellet will dissolve upon heating and the bromophenol blue colour buffer has turned green? Anything I can do or idea's as to why this has happened?

Thank you!

Petra

Petra on Sat Sep 3 18:44:59 2011 said:

Hi Petra.....welcome to bioforum...it would be better if you post your extraction protocol so people here can have an idea of what you did. If your samples are acidic, the colour can change to yellowish/greenish so then you may need to neutralise them first with Tris pH 8.8 or HEPES or even NaOH. Sometimes though, they can turn back to blue after you've loaded them into the wells of your gel. And if you haven't done it already- did you try vigorously pipetting in and out then vortexing when you first re-suspended them in the loading buffer? You can always try sonication to break up the pellet....

Thank you,

This is the protocol I used -I've now used it three times and each time had similar problems (last time pellet not fully dissolved but colour OK, previously no solid material but very green(there was not enough protein in this one I think), however the person who gave it to me seemed to use the same protocol without any problem.

I have made sure I'm not over drying the pellet of acetone so I don't think this is the issue...


Protocol:
<*>Add some glass beads (Sigma, acid washed) to tube (make holes in lid with needle to allow pressure escape) and place on ice.

<*>Grind 1ml of (~7 day) old suspension culture in eppendorf using an eppendorf grinder. Try and keep tube on ice as much as possible during this step. Wash grinders in 0.1M HCl and then dH₂O after use.

<*>IN FUME HOOD, on ice. Add 1ml 0.2M NaOH 2% b-mercaptoethanol, plus 10ml protease inhibitors (for 5ml: 714µl protease inhibitors, 100µl b-mercaptoethanol. 10ml: 1428µl PI, 200µl b-mercaptoethanol) to leaf material in an eppendorf and grind using an eppendorf grinder for a few mins.

<*>Spin 13000rpm 10mins 4ºC


<*>Remove supernatant to a clean 2ml tube and add an equal volume of 80% acetone 20% TCA (tri-chloro acetic acid) solution (prepare fresh), mix and leave on ice for 30 mins minimum (up to 2h).

<*>Spin 13000rpm 10mins, remove supernatant completely and wash pellets twice in 100% acetone (5 min 13000rpm)

<*>Spin 13000rpm 5mins and carefully remove supernatant, pellet of protein will be very loose.

<*>Allow pellet to air dry to remove all of the acetone. Do NOT overdry, only needs a few mins. Leave lid off tube.


<*>Resuspend pellets in 100µl (or 200 if more volume required) 1x SDS sample buffer (premix water and 2x buffer prior to adding), heat 5 mins 95ºC.

the whole thing here is you are using TCA precipitation of proteins:
1. TCA is very very strong acid so if you don't remove it properly you are bound to get change in color from blue to green

and the other thing which is interesting if you using the pellet after breaking the cells which is cell membrane and membrane proteins and they always will give smear on SDS PAGE. (My only concern here is why are you using TCA precipitation for the membrane)

Please check the protocol try doing TCA precipitation for soluble fraction too.

-OR-

dissolve the final pellet in any buffers like 1X PBS and take 10-50µl and then add 1X SDS buffer the smear problem should be gone.

Best Luck