Biouday

It seems that there is no one marker for smooth muscle, you may have to use more than one.  One paper I found recommended SMα-actin, SMC myosin heavy chain (SM-MHC), Calponin, SM22α, and H-caldesmon.

Biouday, on 21 December 2011 - 03:39 AM, said:

Definitely.  The pores in the membranes are much much larger (they are nearly the size of a bacterium) than any proteins you are likely to be detecting on a western.  The liklihood of a protein migrating through depends on the membrane and transfer system you are using.

pakbiochemist, on 21 December 2011 - 11:30 AM, said:

If you loaded only histone proteins, you won't see any other proteins, so why bother trying to detect them?  If you actually loaded 50 ug of total cell lysate (as I suspect), then it may be worth stripping.  Personally I disapprove of stripping, as you don't know how this affects the proteins that are on the membrane, and potentially alters any results you might have, unless you are very careful to keep the stripping conditions consistent.

You can't tell if the transfer has gone correctly, unless you ponceau stained the membrane.  If you have consistently been able to transfer proteins using this method before, then you can be reasonably sure that you will have protein on the membrane.  IIRC histones are likely to be able blow through the membrane as they are reasonably small (approx 15 kDa).

Your stripping will work, but you should determine the number of times you can do it for yourself.  I would estimate that a maximum of 2 times (detect 3 proteins) would be about the limit!

Please give us further information.

How did you set up the reaction?
Which enzymes are you talking about?