Transfer sandwich membranes

The lack of selectivity can be circumvented by coupling a postcolumn flow system to a liquid chromatograph. This has promoted the development of a number of efficient liquid chromatography-CL approaches [16, 17]. Eluted analytes are mixed with streams of the substrate and oxidant (in the presence or absence of a catalyst or inhibitor) and the mixed stream is driven to a planar coiled flow cell [18] or sandwich membrane cell [19] in an assembly similar to those of flow injection-CL systems. Many of these postcolumn flow systems are based on an energy-transfer CL process [20], In others, the analytes are mixtures of metal ions and the luminol-hydrogen peroxide system is used to generate the luminescence [21],... 

The actual blotting process may be accomplished by one of two methods passive (or capillary) transfer and electroblotting. In passive transfer, the membrane is placed in direct contact with the polyacrylamide gel and organized in a sandwich-like arrangement consisting of (from bottom to top) filter paper soaked with transfer buffer, gel, membrane, and more filter paper. The sandwich is compressed by a heavy weight. Buffer passes by capillary ac-... 

Figure B3.2.1 Aligning the gel for transfer. (A) If the polyacrylamide gel has been loaded from left to right, cut a small piece from the corner of the lower left-hand edge of the gel near the first lane. (B) When preparing the transfer sandwich, turn the gel over so that the cut edge is on the lower right-hand corner of the gel. This will ensure that the transferred proteins will appear in the same order as in the original gel. (C) After transfer, trim the membrane above the cut corner of the gel to mark orientation. Dots indicate where the proteins were before transfer.

Do not allow any area of the PVDF membrane to dry during assembly or transfer will not occur in these areas. The transfer sandwich should fit together snugly to provide good contact between the membrane and gel. The complete gel sandwich should look like that in Figure B3.2.2. 

At the end of the transfer period, turn off the power and disconnect the power supply. Open the transfer sandwich and remove the membrane and gel (Fig. B3.2.1). Thoroughly rinse the membrane with high-purity water, three times, 5 min each. 

An alternative to the tank transfer system is the semidry transfer system. In this procedure, the gel is stacked horizontally on top of the membrane in the transfer apparatus. Because only a small volume of transfer buffer is used, SDS from the gel is less effectively diluted, which may result in incomplete binding and lower yields, especially with PVDF membranes. For this reason, semidry transfer units are not recommended when reproducible high recoveries of electroblotted proteins are desired (e.g., for subsequent sequence analysis). Some procedures recommend stacking multiple transfer sandwiches to achieve several transfers simultaneously. To prevent unbound protein from migrating through the next gel and onto the membrane in the next transfer stack, sheets of porous cellophane sheets or dialysis membrane are placed between adjacent transfer stacks (see Fig. B3.2.3). Semidry electrotransfer requires shorter transfer times than tank transfer. 

Figure B3.2.3 Electroblotting with a semidry transfer unit. In most cases, the lower electrode is the anode, as shown. Position the Mylar mask (optional) directly over the anode. Layer on three sheets of filter paper that have been wetted in transfer buffer. For negatively charged proteins, place the preequilibrated transfer membrane on top of the filter paper followed by the gel and three additional sheets of wetted filter paper. If multiple gels are to be transferred, separate the transfer sandwiches by inserting a sheet of porous cellophane or dialysis membrane between each stack. Place the cathode on top of the assembled transfer stack(s). Transfer the proteins by applying a maximum current of 0.8 mA/cm2 gel area.

When assembling this transfer sandwich, the gel is placed on top of the membrane, in contrast to the opposite order for tank type sandwich assembly as in Basic Protocol I. Therefore, it is unnecessary to turn the gel over to obtain the proper orientation (compare Figs. B3.2.1 and B3.2.3). 

Assemble the gel/membrane transfer sandwich and place in transfer tank (see Basic Protocol 1, steps 7 to 10). 

During electrotransfer, proteins migrate out of gels in an electric field according to the charge on the protein. Most electrotransfers employ a tank transfer apparatus (Fig. B3.2.2) in which the gel/membrane transfer sandwich is mounted in a cassette and placed in a tank of Tris/glycine/methanol transfer buffer. The... 

The peripheral equipment needed for direct methanol fuel cells is largely analogous to that of polymer electrolyte membrane fuel cells. The mechanical basis of fuel cells and stacks on the whole consists of bipolar plates between which the sandwiched membrane-electrode assemblies are arranged. For the venting of heat, cooling plates with a circulating heat transfer agent are set up in a particular order between individual fuel cells in the stack. 

Figure 2 Western blot transfer sandwich assembly for wet transfer (top) and the preparation for transfer using the Mini-Trans-blot apparatus (bottom). The transfer membrane is sandwiched between the gel, filter papers, and support pad, with the transfer membrane facing the anode. The cassette containing the assembled gel-membrane sandwich is inserted into the Trans-blot apparatus containing cold transfer buffer, and the electro-transfer carried out. (Adapted from Bio-Rad Instruction manual.)...

Once the sandwich is constructed, place it in the transfer apparatus in a buffer tank that contains lx transfer tank buffer. Make sure the bottom side of the transfer sandwich is toward the negative (black) terminal. Transfer the proteins from gel to membrane using current and voltage settings appropriate for the electrotransfer unit. 

Assemble the transfer sandwich Scotch Brite pad (or equivalent scouring pad), three layers of Whatman 3MM paper, the gel, the membrane (nitrocellulose, nylon, or Immobilon-P—the latter has to be prewetted in methanol), three layers of 3MM paper, and another Scotch Brite pad. Transfer gel-fractionated proteins electrophoretically to a membrane under conditions that have been experimentally determined to be best for the protein of interest. With a Bio-Rad Trans-Blot apparatus, transfer of a 60-kDa protein can be accompUshed using 60 V for 1 hr. 

Figure 2 Composition of a transfer sandwich with plate electrodes at the top and bottom. The polyacrylamide gel is placed onto the membrane, and the assembly is tightly compressed using ScotchBrite sponge pads to ensure ciose contact of the gei and membrane. The transfer sandwich is then submerged in a tank of buffer for electrophoretic transfer of proteins from the gel on the cathode side to the membrane on the anode side.

After transfer is complete, remove the transfer sandwich. The gel can be stained in Coomassie blue to assess efficiency of transfer, and the nitrocellulose membrane may be stained with Ponceau S (Protocol 6) to visualize the total protein pattern prior to immunoblotting (Protocol 7). 

After the initial separation by a conventional electrophoretic technique in a gel, the proteins are transferred (or blotted) electrophoretically from the gel to a membrane, usually nitrocellulose. The gel and the membrane, which has been previously soaked in a suitable electrophoretic buffer, are sandwiched between two electrodes. A voltage is applied, e.g. 100 V, and the proteins migrate from... 

Figure 8.12 Two types of electrotransfer apparatus. At the left a tank transfer cell is shown in an exploded view. The cassette (1) holds the gel (2) and transfer membrane (3) between buffer-saturated filter paper pads (4). The cassette is inserted vertically into the buffer-filled tank (5) between positive and negative electrodes (not shown). A lid with connectors and leads for applying electrical power is not shown. On the right side of the figure is shown an exploded view of a semidry transfer unit. The gel (5) and membrane (6) are sandwiched between buffer-saturated stacks of filter paper (4) and placed between the cathode assembly (3) and anode plate (7). A safety lid (1) attaches to the base (9). Power is applied through cables (8).

In standard basic transfers from SDS-containing gels, the gel should be on the cathode (negative) side of the sandwich, and the membrane on the anode (positive) side of the sandwich. For acidic transfers, the gel and the membrane positions should be reversed... 

Fig. B.5 Cartoon depiction of the Western Biot sandwich. The gei is piaced with the membrane between a set of sponge pad and 3M papers on either side and the transfer is done with the membrane on the negative eiectrode.

It is important to avoid air bubbles when assembling the blotting sandwich cassette in order to allow proteins to be transferred properly to the nitrocellulose membrane. 

Multiple electrotransfer of up to three gels is possible. On top of the filter papers soaked with Soln. B a wetted dialysis membrane is placed and then the sandwich is repeated. Transfer conditions are similar to the single sandwich except the total gel area taken into account for calculation of applied current. 

Figure B3.2.2 Electroblotting with a tank transfer unit. The polyacrylamide gel containing the protein(s) to be transferred is placed on the smooth side of the polyethylene sheet (or filter paper sheets) and covered with the PVDF membrane and then a single sheet of filter paper. This stack is sandwiched between two fiber pads and secured in the plastic gel holder cassette. The assembled cassette is then placed in a tank containing transfer buffer. For transfer of negatively charged protein, the membrane is positioned on the anode side of the gel. Charged proteins are transferred electrophoretically from the gel onto the membrane.

The membrane system considered here is composed of two aqueous solutions wd and w2, separated by a liquid membrane M, and it involves two aqueous solution/ membrane interfaces WifM (outer interface) and M/w2 (inner interface). If the different ohmic drops (and the potentials caused by mass transfers within w1 M, and w2) can be neglected, the membrane potential, EM, defined as the potential difference between wd and w2, is caused by ion transfers taking place at both L/L interfaces. The current associated with the ion transfer across the L/L interfaces is governed by the same mass transport limitations as redox processes on a metal electrode/solution interface. Provided that the ion transport is fast, it can be considered that it is governed by the same diffusion equations, and the electrochemical methodology can be transposed en bloc [18, 24]. With respect to the experimental cell used for electrochemical studies with these systems, it is necessary to consider three sources of resistance, i.e., both the two aqueous and the nonaqueous solutions, with both ITIES sandwiched between them. Therefore, a potentiostat with two reference electrodes is usually used. 

Assemble the sandwich for transfer in this order fiber pad, filter paper, nitrocellulose, gel, filter paper, and fiber pad. Remove all air bubbles between membrane and gel and between paper and gel. 

Figure 4-32. Southern blotting. In Southern blotting, electrophoretically-separated DNA fragments on a gel are transferred by capillary action on to a nitrocellulose membrane. This is achieved by having a tightly packed sandwich of filter paper, gel, nitrocellulose filter, filter paper...

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