Polyacrylamide Gel as a Matrix for the Delivery of a Layer or Coat of Other Molecules

In this work, we demonstrate the potential of thin polyacrylamide gels as a material or matrix for the incorporation of other molecules to deposit or deliver a uniform layer or coat of the molecules for applications unrelated to electrophoresis. Polyacrylamide has been used as a matrix for entrapment of a number of molecules for different purposes (7,9,12). For instance, entrapment of collagen in polyacrylamide granules for the purification of collagenases has been described (12). However, in our knowledge, there is no report of the use of cast gels as thin layers for a coating application or delivery of a molecule in a layer onto another surface. We suggest that polyacrylamide gels can be used for overlaying applications, either wet or in dried form. As an example, in the present work, we have shown that a polyacrylamide gel in the dried form can be used for fluorography of tritiated small molecules. Tritium is a radioisotope that has applications in experiments requiring 3Hlabeled precursors. Film detection of the low-energy β particles from tritium decay is very inefficient by autoradiography because these particles do not penetrate the silver halide photographic emulsion on X-ray film sufficiently (15). So, for detection of tritium-containing molecules, the technique of fluorography (11) was developed (18), in which a scintillant or fluor such as 2,5diphenyloxazole (PPO) (3) is used. Upon application, the scintillants emit light when the β particles from radioactive disintegration impinge on them. Thus, the multiple photons generated by the fluor, rather than the weak β particles from 3H disintegration themselves, cause activation of the photographic emulsion on X-ray films. Thus, for film detection, the energy released by radioactive molecules is converted to visible light by solid-state scintillation (1). For fluorography of thin supports such as paper chromatograms, thin layer chromatography (TLC) plates, or nitrocellulose/nylon filters, the methods developed involve the incorporation of a scintillant into the thin support by dipping into a solution of scintillant (4,10,14) or in melted scintillant (4), or by including a scintillant in the preparation of a thin layer of slurry for TLC (11). Spray formulations of scintillants are also available commercially (NEN Life Science Products, Boston, MA, USA). For fluorography of 3H-labeled molecules in polyacrylamide gels, a method using PPO has been described (3) in which the gel is soaked in dimethyl sulfoxide (DMSO) to replace water and then in a solution of PPO in DMSO. The PPO gets incorporated into the gel; the gel is placed in water so that the incorporated PPO precipitates within the gel matrix. The gel is dried, and X-ray film is exposed to it. Here, an alternative method for fluorography of tritium-containing small molecules on thin filters is described. This procedure, adapted from the method described above for fluorography of polyacrylamide gels, recruits the capacity of polyacrylamide gel to be efficiently embedded with a large amount of PPO and uses a polyacrylamide gel as a matrix for application of PPO to the filter. The protocol followed is outlined below. A 15% polyacrylamide gel mixture was prepared using acrylamide, bisacrylamide, TEMED, ammonium persulfate, and water. No SDS or Tris buffer was added. The solution was mixed thoroughly to ensure uniform lattice formation (important for even PPO incorporation) and poured in a Hoefer vertical gel cast (Hoefer Scientific, San Francisco, CA, USA) using 0.5-mm spacers. A 15% gel concentration was chosen because these gels are easy to handle and do not break easily. The gel was removed from the cast and processed for fluorography as described earlier (2,3). Briefly, the gel was placed in DMSO for 2 × 30 min with gentle agitation. The gel was transferred into a 20% (w/v) solution of PPO (Loba Chemie, Mumbai, India) in DMSO and kept for 2 h with gentle agitation. The PPO-embedded gel was kept under slowly running water for 1 h. For demonstration of this method, 60, 300, 900, 1500, and 2700 dpm/mm2 spots of tritiated thymidine (NEN Life Science Products) were made on Whatman no. 1 filter paper (Whatman, Kent, UK). Dot blot filters were prepared in triplicate, one filter serving as the nonfluorographed control, one to be fluorographed by dipping in a solution of scintillant, and one to be processed according to the method described in this work. The diameter of each spot was 4.5 mm. The disintegrations per minute were determined, taking into consideration the half-life of tritium, and also verified using the Packard TriCarb 2100TR liquid scintillation counter (Packard Instrument, Downers Grove, IL, USA). One filter was dipped into a solution of PPO in toluene (20% w/v) (17) and air dried. The second filter to be fluorographed was placed on a Whatman no. 3 filter paper. The PPO-embedded gel, as prepared above, was cut to a size a little larger than the filter (0.5–1 cm) on all sides, lightly dabbed with tissue to remove excess water, and then this was placed over the filter, the gel extending a little beyond the filter on all four sides. The assembly was covered with cling film, and the gel was dried over the filter for 1 h at 65°C in a Bio-Rad gel drier (Bio-Rad Laboratories, Hercules, CA, USA). Marking the position of the filter is not required because the PPO-embedded gel becomes transparent upon drying and the paper becomes completely visible through the gel. The cling film was removed, and X-ray film was exposed to this filter over which a layer of PPO had been applied using a polyacrylamide gel. Thus, a PPO-embedded polyacrylamide (PEP) gel screen was placed over the filter before exposure. The film was exposed at -70°C. The filter that was not fluorographed and the dipped filter were placed, devoid of any wrap, next to the filter with PEP screen and autoradiographed on the same piece of X-ray film for the same time. The X-ray film was developed for 5 min at 25°C–28°C and fixed. Kodak X-Omat AR film (Eastman Kodak, Rochester, NY, USA), which is highly sensitive to the blue light emitted from PPO (6) was used without preflashing (8). Kodak DA-163 developer (stock solution without dilution) was used. The results are shown in Figure 1A. No signal was visible from the filter that was not fluorographed (not shown Benchmarks