Blot Name Conventions (was Probing native DNA on filters)

mangalam at SALK-SC2.SDSC.EDU mangalam at SALK-SC2.SDSC.EDU
Thu Sep 24 13:18:40 EST 1992


Ed Rybicki writes:

>Southern blotting: blotting of denatured DNA onto membrane, probe with
>    DNA or RNA
>
>Northern blotting: blotting of denatured RNA onto membrane, probe with
>    RNA or DNA
>
>Western blotting: blotting of denatured protein onto membrane, probe with
>    antibody
>
>Southern Cross blotting: blotting of 1 wide lane of restricted DNA onto a
>    filter, probe with another single-lane digest of end-labelled DNA from
>    a gel turned at right-angles to the first (to detect common sequences
>    between fragments in different digests)
>
>Middle Eastern blotting: blotting of RNA onto membrane, using it as
>    substrate for in vitro translation, detection of translation product
>    using antibody
>
>South-Western blotting: getting drunk on Hansa Pilsener [local joke:
>    Namibia used to be called SOuth_west Africa, and they make very good
>    beer there...!]

While I enjoy Ed's def'n better, mine is:

SouthWestern blotting: blotting of protein to a membrane, then probing with
labelled DNA.  Depending on the protein, you may be able to subject the
blot to denaturation/renaturation to eliminate the activity of confounding
proteins.  A tremendously useful way to characterize DNA-binding proteins. 
I have a particular fondness for this technique as it was the breakthru
that allowed me to do finish my thesis work 8) and contributed to our lab
cloning some very interesting transcription factors.

Uh Oh, I feel a flame coming on..... I can't stop it!!!  Watch out.......

{Flame on}
    I'd like to slide a snide side diatribe in:  As one who has watched,
with dismay and disgust, the proliferation of gel shifts (aka
(appropriately) gel retards) being presented in Denatured, Cool, etc (ie:
the BIG CHEESE journals), I'd like to say that anyone who has worked the
protein side of the fence should know how inaccurate these things can be,
especially when done with _unfractionated_ cellular or nuclear extracts.
   Merely because band shifts migrate to the same position in gels doesn't
mean the proteins are the same.  Especially, when the bands are being
compared from different gels, relative to other bands (!!!)  Or from
different cells (!!!)  Or to assume that because a band migrated higher in
the gel, it's a larger protein (!!!)  I was burned many times early on by
assuming these points and it burns me more to see this kind of oleaginous
data greasing the slides for yet another BIG (and litle) CHEESE paper to
careen into the literature, only to have it shown years (and thousands of
hours and dollars) later that the data was incomplete or simply incorrect.
   Unless the data can be validated by (several) other methods, I'd like to
see gel shifts banned by international convention 8) (but just barely).
{Flame off}

   Sorry, but I feel better now...

Cheers,
Harry

Harry Mangalam                                   Vox:(619) 453-4100, x250
Dept of Biocomputing                                   Fax:(619) 552-1546
The Salk Institute                             mangalam at salk-sc2.sdsc.edu
10010 N Torrey Pines Rd                        mangalam at salk-sgi.sdsc.edu
La Jolla CA 92037                                    mangalam at salk.bitnet




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