3 replies to this topic

[ashhbel]

Hi,

I attempting to to a western blot for the NLRP3 protein which makes up part of the inflammasome, however I am having problems with this

My samples are macrophages which have been cultured for 7 days in MCSF and GMCSF.  These cells are then stimulated with LPS and ATP

After this the cells are lysed in m-PER(Thermo) and proteinase and phophatase inhibitors added (however these were frozen and may be out of date).  The protein is then stored at -20 in acetone.  Once precipitated I dissolved the proteins in 1x laemlli buffer to make a concentration of 3µg/ml, heated for 5mins and stored at -20

I have been attempting to run the samples on a 4-12% NuPAGE Bis-Tris Gel.  However the samples do no appear to run correctly, but as a frown and then when the blot was treated with ponceau the bands in some lanes are very thin width wise

I was just wondering if anyone could suggest why this is happening as I have ran a few times with different samples and get the same result

I have attached the blot ponceau stained

Thanks

Ash

Attached Thumbnails

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[bob1]

Are you sure the gel that you are running them on is denaturing?  I have seen patterns like that for native gels.

[mdfenko]

are you sure that the pellets are free of acetone?

Edited by mdfenko, 19 April 2012 - 12:05 PM.

[Inmost sun]

maybe some lipids are left in your sample which cause the streaks; as mdfenko asks, traces of acetone may contain lipids...