myosin labelling on c2c12 cells

Michael Dorsett Onken mdonken at artsci.wustl.edu
Thu Oct 29 10:52:31 EST 1998


Qunfeng Dong (qfdong at iastate.edu) wrote:

: Hi, has anyone here tried to use MF20 to label fused c2c12 cells before?
: I always get non-specific labeling. I used 2% paraformaldehyde for 3 mins,
: then use 0.5% TritonX-100 for 1 min. Then either block with 0.5% BSA or
: without blocking for 30 mins. Label 1st Ab for 1 hr and 2nd Ab for 1. I
: tried different wash time, either 5 mins each for 3 times or 30 sec each for
: 3 times. Anyway, I never get good specific labelling on myofibril or those
: stress-fiber like structure. Please help. I need to know your detail
: labelling procedure. Please send me an personal email. I am not regular
: reader here. Thanks a lot and have a nice day!

: --Qunfeng
: -- 
: Qunfeng Dong
: qfdong at iastate.edu

We've had the same problems with various anti-myosin antibodies on C2C12
tubes, and have found that switching to My32 (from Sigma of all places)
greatly increased our signal to noise ratio.  The only changes we've made
to the above protocol are: 6' -20C MeOH instead of 1' Triton-X100; and 2%
"fish gel" (teleostean gelatin - also from Sigma) instead of 0.5% BSA.
Ultimately, it was the antibody that made the difference, and now our
staining works beautifully.  As an aside, I never thought I'd see the day
I quoted Sigma twice in one breath.  Oh well, at least it wasn't
VectaStain...

--
Mike Onken        -. .-.   .-. .-.   .  E-mail:
Molecular Cell    ||X|||\ /|||X|||\ /|   mdonken at artsci.wustl.edu
Biology Program   |/ \|||X|||/ \|||X||  URL:
Washington Univ.  '   `-' `-'   `-' `-   http://madsci.wustl.edu/




More information about the Cellbiol mailing list