Semi-dry blot buffers

marder at agri.huji.ac.il marder at agri.huji.ac.il
Sun May 16 02:14:48 EST 1993


In article <1993May12.131842.1 at agri.huji.ac.il>, marder at agri.huji.ac.il writes:
> Hi,
>   I am a user of a semi-dry electrophoretic blotter (Biorad model) for
> doing protein transfers from gel to nitrocellulose.    Basically, things
> work, but it seems our transfer efficiency is less than for wet blots
> (increaseing the time can get more protein out of the gel, but not
> necessarily more on the NC).   etc.

In addition to the replies that appeared in this newsgroup, I also received
a few others by private email (appended below).  Thanks to all who replied.

Message-ID: <MAILQUEUE-101.930512064333.566 at micro.uct.ac.za>
To:           marder at agri.huji.ac.il
From:         "Ed Rybicki"  <ED at micro.uct.ac.za>
Date:         12 May 93 06:43:33 SAST-2
Subject:      Re: Semi-dry blot buffers
Reply-to:     ed at micro.uct.ac.za
Priority:     normal
X-mailer:     Pegasus Mail v2.3 (R4).

THIS COMES STRAIGHT OUT OF A METHODS MANUAL COMPLIED BY ED RYBICKI,
DEPT MICROBIOLOGY, UNIV OF CAPE TOWN; REFERENCE HIM AS PERSONAL
COMMUNICATION OR FIND REFERENCE ATTACHED TO METHOD.



                                               PROTEIN MOLECULAR METHODS 1


                     .I.HORIZONTAL ELECTROBLOTTING BLOTTING:
                                  FOR PROTEINS;

        By: E.P. Rybicki, Dept. Microbiology, Univ. Cape Town.

        INTRODUCTION
        Electroblotting has been a feature of a large number of
        laboratories for a (relatively) long time now, and there are a
        large number of different apparati around that will efficiently
        transfer proteins (or other macromolecules) from gel to membrane.
        Most of these, however, are based on the design of Towbin et al.
        (1979): ie., they have vertical carbon / stainless steel /
        platinum electrodes in a large tank.  Some enterprising newcomers
        have fairly recently turned the whole issue on its side, and have
        started "semi-dry" or "horizontal" blotting.  With this
        technique, one uses two plate electrodes (stainless steel or
        graphite / carbon) for uniform electrical field over a short
        distance, and sandwiches between these up to six gel / membrane /
        filter paper assemblies, all well soaked in transfer buffer.  The
        assembly is clamped or otherwise secured ON ITS SIDE, and
        electrophoretic transfer effected in this position, using as
        transfer buffer only the liquid contained in the gel and filter
        papers or other pads in the assembly.  There are a number of
        advantages to this procedure over the conventional upright
        protocol, not the least of which is that as little as a couple of
        hundred millilitres of buffer are all that is needed for
        electroblotting several gels, compared to five or so litres
        for some commercial kits.  In addition, several gels can be
        blotted simultaneously; electrodes can be cheap carbon blocks;
        less power is required for transfer (and therefore a simpler
        powerpack).

        PROTOCOL
        We have simply adapted our previous "poor man's" vertical set-up
        for horizontal use, making use of nappy liners as absorptive
        buffer-containing pads.  The procedure consists of making an
        assembly of two 1-cm-thick carbon electrodes on either side of
        two wads of nappy liners, with as many as six series of filter
        paper/gel/membrane/filter paper stacks inside the nappy liner
        pads (see Figure). NOTE FOR YANKS: NAPPY=DIAPER
        Equipment
        1)   Two carbon block electrodes (eg. 20x15x1 cm) with heavily-
             insulated wire connectors, soldered into a hole drilled into
             the block then cemented in place (Le Carbone Ltd., Cape Town
             - brushes for electric motors; +R120-00/pr).
        2)   Destainer power pack or equivalent: the Shandon Southern
             powerpack gives 1A at 50V; you can get away with 500mA and
             25V or so.
        3)   1 pack nappy liners, fine weave (cut to size).  Make sure
             these are highly absorbent and wettable.  They may be re-
             used endlessly.  Sustitute cheap absorbent non-
             disintegrating material if preferred.
        4)   Whatman No. 3 filter paper or plain blotting paper - it's a
             lot cheaper, as you throw it away after one use.
        5)   Nitrocellulose or other adsorptive membrane: nitrocellulose
             is OK for adsorption and gives low staining backgrounds with
             India ink, Amido Black, Coomassie, colloidal gold and

                                               PROTEIN MOLECULAR METHODS 2


             peroxidase and alkaline phosphatase substrates but is
             fragile; Amersham make a tough NC which may be more
             suitable.  They also make Hybond-N (nylon) which is
             amazingly strong but used to give horrible background with
             enzyme-immune substrates; the Boehringer non-radioactive
             nucleic acid detection kit blocking agent gets around this.
             No nylon can be stained with the other stains; however,
             Millipore claims their polyvinylidene difluoride (PVDF)
             membranes are as good as nylon and can be stained with gold.

        Buffers
        1)   Blotting buffers: there are several choices here.
          a) We use the simple Towbin buffer (50mM Tris/196mM glycine/20%
             methanol, pH 8.3) from habit.  It may be an idea to reduce
             the conductivity of this buffer so as to allow a slightly
             higher voltage for transfer: cut molarities by half or more.
             Leave the methanol at 20% (v/v): it is there to stop the gel
             swelling due to heating, and to keep proteins denatured for
             attachment to the membrane.
          b) It is also possible to use 0.025 or 0.1M phosphate pH
             7.0/20% MeOH - haven't tried it personally.
          c) My German friend Jorge Meyer (Botanisches Institut, Aachen)
             writes that one may also use anodal and cathodal buffers as
             follows:
                  Anodal 1 (for pad nearest anode): 0.3M Tris/20%MeOH pH
                       10.4
                  Anodal 2 (for other gel pad assemblies): 25mM Tris/20%
                       MeOH pH 10.4
                  Cathodal (for all pads etc. nearest cathode): 25mM
                       Tris/40mM aminocaproic acid/20% MeOH, pH 9.4.

        Procedure
        1)   Fractionate proteins by SDS-PAGE or other gel procedure;
             soak gel in transfer buffer of choice for 10 - 60 min.
        2)   Soak membrane, and as many single sheets of blotting paper
             as needed (two per gel assembly), and as many pads of nappy
             liners as required (eg. I use two: a 1-cm pad at the
             cathode, and a 1.5-cm pad at the anode) in chosen buffer(s).
        3)   Put cathode in a shallow tray (on a plastic bag/sheet), and
             add soaked cathodal buffer pad, and all the soaked filter
             paper, gel, membrane, filter paper assemblies (space these
             with a thin pad of soaked nappy liner if wished/required),
             add anodal nappy liners and cover with anode.
        NOTE: AVOID BUBBLES AT ALL TIMES - CARRY STACKS, PADS CAREFULLY
             SO AS TO AVOID BUFFER RUNNING OUT OR DEFORMATION LEADING TO
             DISPLACEMENT OF LAYERS.
        4)   Put a weight of some kind on anode: a 250ml reagent bottle
             (empty) should be more than enough.  Cover whole assembly or
             erect a warning sign:  APPARATUS OPERATES AT HIGH CURRENT
             LEVELS!  BE CAREFUL WHEN SWITCHED ON!!!
        5)   Attach leads to powerpack: run at 0.5 - 1A/300 cm2 for 30 -
             60 min.  Thin assemblies take very low voltage, so maybe
             reduce buffer ionic strength for single-gel transfers.  One
             hour should be sufficient for transfer of all but the most
             recalcitrant proteins.  If in doubt, blot gels in duplicate,
             remove one and stain for protein after 1 hr and continue
             blotting other.

                                               PROTEIN MOLECULAR METHODS 3


        NB: ALWAYS KEEP SAME ELECTRODES AS CATHODE AND ANODE - THEY WILL
             FLOOD YOUR SYSTEM WITH COLLOIDAL CARBON IF YOU REVERSE THEM
        6)   Disassemble, saving nappy liner pads (put straight back into
             buffer on tray if to be re-used soon).  Rinse membrane in
             saline or other buffer before further treatment.

        NB: CAN USE THE SAME ASSEMBLY FOR BLOTTING NUCLEIC ACIDS - JUST
             CHANGE BUFFERS TO ONES APPROPRIATE FOR NUCLEIC ACID (eg.
             0.025M phosphate pH 6.5 for DNA or RNA; see Northern
             electroblot protocol).



                  .I.ELECTROPHORETIC BLOTTING OF dsRNA OR DNA;

        Author: E.P. Rybicki, Dept. Microbiology, UCT.

        From: Susan Hall, Hons. thesis (1986): "The use of double-
             stranded RNA in the identification and characterisation of
             cucumber mosaic virus".

        NB: Electrophoresis can be in either agarose or polyacrylamide.
        Recipe is for agarose - reduce incubation times by up to 50% for
        thinner polycrylamide gels if required.

        1.   After electrophoresis in non-denaturing gel, soak gel in
             0.5-1.0 ug/ml EthBr for 5-10 min.  View on UV
             transilluminator, usually 254 nm, preferably on sandwich
             wrap (to prevent contamination).  Preferably with light off,
             cut away excess gel (sides and ends).

        2.   Denature RNA gel by soaking in 50mM NaOH for 30 min at room
             temperature with gentle shaking.  Denature DNA in 0.5M NaOH,
             not necessarily with high salt.

        3.   Cut Hybond-N/nitrocellulose and 2-6 (see below) sheets of
             Whatman 3MM paper to same size as gel piece, and soak in
             transfer buffer (0.025M phosphate pH 6.5).

        4.   Neutralise RNA gel by soaking in transfer buffer for +30
             min: DNA gels may have to be neutralised better than this.

        NB: SOAKING VOLUMES SHOULD BE +10X GEL VOLUME; IF LESS, CHANGE AT
             LEAST ONCE.

        5.   Prepare cassette of Hoefer Transphor, or of "Burger's Baby"
             mini-blotter, by placing 3 sheets paper on sponge, overlay
             with membrane, and overlay with gel.  Cover gel with 2-3
             sheets of paper, and assemble cassette and insert carefully
             into blotting tank.


             ALTERNATIVELY:
             prepare for horizontal blotting exactly as outlined in the
             protocol, using the appropriate buffer for nucleic acid
             transfer.

                                                   RNA MOLECULAR METHODS 4


        NB. ENSURE NO BUBBLES TRAPPED AT ANY STAGE: THESE INTERFERE WITH
             TRANSFER.

        6.   Ensure transfer membrane is nearest the anode.
             Electrophorese at 0.5-1.0 amps (Shandon Southern destain
             powerpack) with fan blowing for 1.5-4.0 hrs for vertical
             transfer; 30 - 60 min. for horizontal.

        7.   Remove membrane carefully, rinse in transfer buffer to
             remove gel fragments (esp. with agarose gels!!), blot dry
             with Whatman 3MM paper.

        8.   Fix RNA or DNA with exposure of (NYLON MEMBRANE ONLY!!)
             membrane to UV on transilluminator for 2-5 min: cover light
             with smoothed-down sandwich wrap , put membrane, RNA side
             down, on light and cover with book to press it down.  300 nm
             wavelength is recommended by Amersham for Hybond-N.

        9.   Store blot sealed on paper with sandwich wrap, or pre-
             hybridise immediately.

        CAN BE USED FOR ss- OR dsRNA BLOTS OR DNA: RESOLUTION EXCELLENT,
                      ESPECIALLY FROM POLYACRYLAMIDE GELS.



                          NEW PROTEIN BLOTTING PROTOCOL

        By: EP Rybicki, M Lynn and S Lindsay, Dept Microbiology, Univ of
             Cape Town  (08/06/1989)

        INTRODUCTION
        Immunoelectro- or Western blotting starts with the
        electrophoretic transfer of proteins separated by gel
        electrophoresis, onto an adsorbent membrane such as
        nitrocellulose.  This transfer may be performed most conveniently
        by horizontal blotting (SEE HORIZONTAL BLOTTING) using nappy
        liner pads soaked in buffer and two large graphite electrodes.
        There have, however, been a number of problems with the
        powerpacks in the Department, as people tend to use thin stacks
        of nappy liners: this leads to a small inter-electrode distance,
        and consequently, a low voltage across the stack.  This means
        that the system is effectively a short-circuit across the
        terminals of the powerpack, and this then heats up alarmingly,
        and blows fuses or burns out.  There is a simple solution to this
        problem:

             REDUCE BUFFER IONIC STRENGTH TO INCREASE RESISTANCE!

        It turns out that most people use the Laemmli running buffer
        formulation of 20% methanol/25-50mM Tris/192mM glycine pH 8.3,
        simplly because recipes say to do so.  There is no good reason to
        use this buffer: it is necessary for good separation in SDS-PAGE,
        but not for transverse electrophoresis.  It is quite adequate to
        use 25mM Tris-Cl pH 8.3/20% methanol (or pH 7.0 - 8.5); for that
        matter, one may equally well use 25 - 50mM phosphate/methanol pH
        7.0 - 8.0.  It is a good idea to use pH values above 7.0, in

                                           PROTEIN MOLECULAR METHODS  5


        order to ensure that all proteins migrate towards the anode.  The
        methanol is necessary to prevent gel swelling with heating, and
        to keep proteins adsorbed to the membrane.

        It turns out that blotting big gels (eg 14.5 x 11 cm) is quite
        adequately done (even for two or more stacked gels) at 0.3 A for
        60 min or less: the system does not heat up appreciably, and
        transfers are good.

        NOTE: you should cut nappy liners and papers to the size of the
        gel(s) to be blotted, so as to minimise surface area exposed to
        the electrode: this reduces the amount of current that needs to
        be passed in order to effect transfer (and incidentally reduces
        heating effects).
  ____________________________________________________________________
 | Ed Rybicki, PhD             |       "Lord, won't you buy me        |
 | (ed at micro.uct.ac.za)        |                                      |
 | Dept Microbiology           |         A Mer-ce-des Benz..."        |
 | University of Cape Town     |                                      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |           - Janis Joplin             |
 | fax: 27-21-650 4023         |                                      |
  --------------------------------------------------------------------



Date: Thu, 13 May 93 17:17:54 EDT
From: plc at med.unc.edu (Philip L. Carl)
Message-Id: <9305132117.AA09054 at cahaba.med.unc.edu>
To: marder at agri.huji.ac.il
Subject: Re: Semi-dry blot buffers
Newsgroups: bionet.molbio.methds-reagnts
In-Reply-To: <1993May12.131842.1 at agri.huji.ac.il>
Organization: UNC-CH School of Medicine
Cc:

We have had excellent results with the 3 buffer system sescribed by J.
Kyhse-Andersen (1984), J. Biophys. Methods 10: 203-209.  Nothing else
seems
to work as well.  The procedure can probably be obtained from Sartorius
c/0 Vangard International 1-800-922-0784


  Phil Carl
  :

  :
Date: Fri, 14 May 93 17:16:30 -0700
From: gelfand at cgl.ucsf.EDU
Message-Id: <9305150016.AA04599 at socrates.ucsf.EDU>
To: marder at agri.huji.ac.il
Subject: Re: Semi-dry blot buffers
Newsgroups: bionet.molbio.methds-reagnts
References: <1993May12.131842.1 at agri.huji.ac.il>


Try the method of Khyse-Andersen (J. Biophys. Biochem.
Methods, vol. 10, pp. 203-209, 1984) - it works great
because it is discontinous versus continous used in
the regular (wet) transfer protocol.

If you do not have the journal, that is basically
what we do:

3 layers of filter paper in 0.3 M tris with 20% MeOH (bufer A)
6 layers of filter paper in 25 mM tris with 20% MeOH (bufer B)
nitricellulose filters wetted in water
gel
6 layers of filter paper in 25 mM tris, 40 mM aminohexanoic acid
(epsilon-amimohexanoic acid) and 20% MeOH (bufer C)
transfer for 1 hr at 250 mA (15 v)

Good luck.

Volodya



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