6 replies to this topic

[Jay Em]

Hi everyone!
I made nuclear and cytoplasmic lysates and used them in western blot. The tubulin control for the cytoplasmic lysates worked beautifully. The Lamin control for the nuclear lysates didn't work at all. This has happened several times now...and I don't understand why.
I'm using the Lamin A/C Antibody #2032 from Cell Signaling for lysates from bone marrow derived dendritic cells. The secondary is anti-rabbit and is working well. (I know this because I'm using it for the tubulin ab as well).
My Nuclear and cytosolic prep protocol:
from Haspel and Darnell (1999; PNAS)

• Lyse cells on ice in buffer A, mix gently by pipetting up and down
• Spin
• Transfer supernatant into a fresh tube (cytosolic fraction)
• Rinse pellet with 1 ml of buffer A
• Spin - remove as much liquid as possible
• Resuspend pellet in equal volume of buffer B
• Freeze @ -80C
• Let samples thaw on ice
• Spin
• Transfer supernatant into a fresh tube (nuclear fraction)

Buffer A

20 mM HEPES pH 7.9
10 mM KCl
1 mM EDTA
10 % glycerol
0.2 % NP-40
1 mM DTT

Buffer B
20 mM HEPES pH 7.9
10 mM KCl
1 mM EDTA
20 % glycerol
420 mM NaCl
1 mM DTT
I use ready made gradient gels from Invitrogen but also tried self-made ones. Always the same outcome...now I'm thinking of using HDAC as a control..
Any suggestions why the lamin isn't working? And any other good ideas for a different nuclear loading control besides lamin and HDAC????
Thanks in advance!

Edited by Jay Em, 28 January 2009 - 07:55 AM.

[little mouse]

did you try laminin detection on your whole extract?

[Jay Em]

little mouse, on Jan 28 2009, 06:10 PM, said:

did you try laminin detection on your whole extract?

Unfortunately I didn't. I don't have whole cell extracts....since I needed as much nuclear as possible...but since I absolutely need the nuclear lysates I need an internal loading control..no matter what..

[Ihla]

I have great luck with Sigma's Beta Actin antibody with our nuclear extract samples. The blot is always very clean, and the signal is strong. Good luck!

[Jay Em]

Ihla, on Jan 29 2009, 07:40 AM, said:

I have great luck with Sigma's Beta Actin antibody with our nuclear extract samples. The blot is always very clean, and the signal is strong. Good luck!

Reviewers do not accept actin as a nuclear loading control as far as I know..

[rkay447]

Look at abcam for loading control antibodies.  They specifically offer TATA binding protein as a nuclear loading control.  I am doing essentially the same experiment but seperating the chromatin from buffer B and looking at chromatin bound proteins versus nucleosolic.  I'm using MCM proteins as a loading since it's been published (and I repeated) that my experimental condition does not disrupt MCM loading and expression is not cell cycle regulated.

[laurequillo]

Use anti HDAC-1 as a nuclear control