Improvements in polyacrylamide gel slicing

ANALYTICAL

BIOCHEMISTRY

Improvements

78, 106- 111 (1977)

in Polyacrylamide

Gel Slicing1

ROY E. GINGERY North Central Region, Agricultural Research Service, U.S. Department of Agriculture, Department of Plant Pathology, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 Received July 8.1976; accepted October 22, 1976 An improved polyacrylamide gel slicer has been devised that provides rapid uniform slicing with a precision of 4-6%. The advantages of this type of slicer are: The gel is sliced directly from the electrophoresis tube; gel diameter and length can vary with no modification of the system; and gels with a range of acrylamide concentrations can be fractionated with no pretreatment of the gel.

Several devices for fractionating cylindrical polyacrylamide gels have been described (I-5) in which the gel is advanced in uniform increments through an opening. After each advance, the extruded portion of the gel is sliced off. The advantages of these systems are simple construction, rapid operation, section uniformity, adaptability to a range of acrylamide concentrations, and no requirement of prior gel treatment such as embedding or hardening through cooling. Ley (6) increased the flexibility of section thickness by using a multiple speed plunger to advance the gel. The limitations of this approach result primarily from the physical properties of the gels. The gel can adhere to the groove or tube through which it is being moved, particularly at low acrylamide concentrations, and gel properties determine the success of the slicing operation itself, particularly if thin sections are being removed. Also, in the devices described (l-6), except for a miniature gel (0.14-cm diameter) slicer (4), removal of the gel from the electrophoresis tube and placement of it into the transporting mechanism increased the opportunity for handling problems and gel distortion. The simplified sectioning procedure described here retains the desirable features of previous systems and offers these additional advantages: A range of gel diameters and lengths can be accomodated without significant modification: all gels within this range are sectioned directly from the tube in which they were electrophoresed; greater flexibility of section thickness is possible; and ease of slicing and transport are improved by alteration of the physical properties of the gels. r Cooperative investigations, North Central Region, Agricultural Research Service, U.S. Department of Agriculture and Department of Plant Pathology, Ohio Agricultural Research and Development Center. Pubhshed with approval of the Director of the Ohio Agricultural Research and Development Center as Journal Article No. 19-76. 106 Copyright All rights

0 1977 by Academic Press. Inc. of reproduction in any form reserved.

ISSN CKQ3-2697

POLYACRYLAMIDE

GEL SLICER

107

FIG. 1. Sectioning apparatus. (A) Overall view of apparatus: (a) gel tube in position in microtome; (b) metal adapter to accomodate tubing connected to gel tube. (B) Close-up view of microtome with gel tube in position.

METHODS Construction and operation. An ISCO (Instrumentation Specialties Co., Lincoln, Nebr.) Model 640 density gradient fractionator2 was modified to drive a IO-ml glass syringe so that slow pumping rates (down to 0.056 ml/mm) could be obtained (Fig. 1A). Tubing was attached to the syringe; the syringe and tubing were filled with water, and the other end of the tubing was placed over the bottom of the gel tube. The gel previously had * Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

108

ROY E. GINGERY

been loosened from the tube by squirting water between the gel and tube with a fine needle syringe. The tube was placed in a laboratory microtome (Faust Scientific Supply, Madison, Wis.) so that the razor blade just cleared the top of the tube during the slicing operation. Free movement of the gel in the tube, essential for accurate sectioning, was easily checked by squeezing the tubing slightly and observing the gel. A small amount of glycerol was used to lubricate the gel, if necessary. The top of the gel was positioned slightly below the top of the tube when the pump was started so that the gel would be transported smoothly at the time of emergence. At the desired time intervals, the gel was sliced, and the section was transferred with a small spatula to a scintillation vial. Section thickness could be controlled by variation of the pumping speed and/or the time interval between slices. The practical lower limit of sectioning was about 0.25 mm with a 6-mm diameter gel. A smaller syringe or a larger diameter gel would allow thinner slices, although 0.25 mm was about the thinnest slice that could be cut accurately from the best gels. Each edge of a razor blade was used for only one gel. Gels for these experiments were cast’in quartz tubes because, in many applications of gel electrophoresis, the gel is scanned with ultraviolet light prior to sectioning. In about 10 min, a 6-cm gel could be fractionated into l-mm sections and the slices could be placed in scintillation vials. Gel preparation. Concentrated 30% acrylamide and 2% bisacrylamide solutions, 3% agarose (which had been refluxed for 15 min), 10x buffer E (0.36 M Tris, 0.18 M sodium acetate, 0.01 M Na,EDTA, 1.8% sodium dodecyl sulfate, pH 7.2), and water were held at 65°C in a water bath. Appropriate amounts of each were mixed to give the desired final gel concentration in 1X buffer E. Ammonium persulfate (10%) and tetramethylethylenediamine were added in the amounts of 0.2 ml and 0.005 ml, respectively, per 24.8 ml of gel solution. The mixture was pipetted into 6-mm diameter quartz tubes and allowed to polymerize at room temperature. Gel tubes were sealed, and the gels were sectioned the following day. RESULTS Performance

The precision of sectioning was tested by slicing uniformly-labeled gels. [3H]Uridine was incorporated into gels during casting, and sections of various thickness were removed. At least 30 slices were made at each setting. Slices were placed in the bottom of a small scintillation vial, covered with 0.5 ml of NCS:water (9:l) (AmershamBearle, Arlington Heights, Ill.) and capped. The mixture was incubated at 50°C for at least 2 hr and allowed to cool. Three milliliters of scintillation cocktail (5 g of PPO, 0.25 g of dimethyl POPOP/liter of toluene) were added, the contents were mixed, and the radioactivity was then measured in a

POLYACRYLAMIDE TABLE SECTIONING

Section thickness” (mm)

Pumping rate (mlimin)

0.87 0.78 0.52 0.52 0.35

0.14 0.084 0.084 0.056 0.056

u Section thickness =

109

GEL SLICER I PRECISION

Slicing interval (set) IO I.5 IO I5 IO

Mean W-4

Standard deviation (%)

16,609 14,962 9,329 9,454 6,339

4.2 3.6 5.1 4.3 5.5

2 X pumping rate X slicing interval 3~ x (gel diameter)2.

Beckman LS-133 liquid scintillation spectrometer (Beckman Instruments, Inc., Palo Alto, Calif.). The results in Table 1 were obtained from a 7.5% acrylamide gel (5% cross-linking) containing 1.5% agarose. The standard deviation in counts per minute among comparable sized sections averaged about 4.5% over the range of section thicknesses, and the average counting efficiency was 46.5%. Sectioning gels of other composition gave comparable results if the lower sectioning limits (see Table 2) were not exceeded. Effect of Gel Composition

on Sectioning

The physical properties of polyacrylamide gels can be altered by variation of the amount or type of cross-linking agent and/or by incorporation of another gelling agent, such as agarose, into the gel. Agarose has been used most frequently in gels of less than 5% acrylamide. The amount of cross-linking and concentration of agarose had significant effects on gel slicing. Table 2 gives the minimum acceptable section thickness for gels of various compositions. Slicing was deemed acceptable if it was easily accomplished for 10 or more consecutive sections that were visually uniform. Concentration of acrylamide, amount of cross-linking, and concentration of agarose all influenced the success of gel slicing. Slightly crosslinked gels without agarose were sticky at low acrylamide concentrations and crumbly at high acrylamide concentrations. Neither sticky (2.5% total acrylamide, 2.5% cross-linking) nor crumbly gels (7.5 and 10% total acrylamide, 1% cross-linking) were satisfactorily sectioned at any thickness. In general, when the amount of cross-linking or agarose was increased, gels could be sliced more easily; agarose concentration had a greater effect on slicing than the amount of cross-linking at all acrylamide concentrations tested. Gels with both high cross-linking and high agarose

110

ROY

E. GINGERY TABLE

EFFECT

OF GEL COMPOSITION Total 2.5

Agarose (%)

2.5

0.0 0.5 1.0 1.5

-c 1.0 I.0 0.52

2

ON MINIMUM

acrylamide

concentration”

5.0

0.83mm 0.78 0.78 0.65

THICKNESS

(%) 7.5

Bisacrylamideb 5.0

SECTIONING

10.0

(%)

10.0

2.5

5.0

10.0

1.0

2.5

5.0

1.0

2.5

5.0

1.0 1.0 0.78 0.43

1.0 0.78 0.65 0.43

0.78 0.65 0.52 0.35

0.78 0.65 0.43 0.35

0.52 0.52 0.52

0.78 0.43 0.28 0.28

0.65 0.31 0.21 0.21

0.78 0.78 0.52

0.65 0.43 0.31 0.28

0.43 0.43 0.35 0.28

D Percentage of acrylamide b Percentage of bisacrylamide c Unable to slice.

+ percentage of bisacrylamide. = bisacrylamide x lOO/acrylamide

+ bisacrylamide.

content were the best. Both agarose and cross-linking appeared to alleviate stickiness at low acrylamide concentrations and crumbliness at high acrylamide concentrations. Gels containing agarose also slid more easily in the quartz tubes than similar gels without agarose. Apparently, swelling was reduced in gels containing agarose. Gels which still slid with difficulty (high acrylamide concentrations) could be transferred easily to tubes with slightly larger inside diameters and then sectioned. DISCUSSION

The criteria for satisfactory sectioning with any gel slicing device are uniformity of sections, ease of operation, flexibility in handling a range of gel types and sizes, and simple control of section thickness. The improvements in sectioning described here satisfy these criteria by the use of the microtome for sectioning, transport of the gel hydraulically by a variable-speed syringe pump, and the incorporation of relatively high concentrations of agarose into the gels. The consistent cutting stroke obtained with the microtome compared to that obtained by using a hand-held scalpel or razor blade resulted in greater uniformity and permitted thinner slices. The slicing stroke was rapid enough to allow continuous pumping during slicing, which further assured steady gel transport and uniform sections. The use of a microtome for sectioning gels has been previously reported (5,7-9); however, gel preparation (cooling or embedding) prior to sectioning was usually required; and, in all cases, the gel had to be removed from the electrophoresis tube. With the microtome described here, the gel, with

POLYACRYLAMIDE

GEL SLICER

111

essentially no preparations, can be sectioned in the electrophoresis tube immediately after running. Hydraulic transport of the gel makes this system more versatile than those requiring plungers or other specialized components, since a range of gel diameters and lengths can be accomodated with no modification. As previously pointed out by Ley (6), the multiple-speed feature permitted simple control of section thickness. The syringe pump on the Model 640 density gradient fractionator could, of course, be replaced by any multiplespeed syringe drive capable of delivering accurate low pumping rates. The incorporation of high concentrations of agarose at all gel concentrations was a key factor that allowed a range of gels to be sliced thinly successfully. Agarose not only helped overcome the difficulties in sectioning gels of low and high acrylamide concentration, but also allowed a greater range of gels to be transported directly in the quartz tubes. Since the amount of cross-linking and the concentration of agarose influence electrophoretic mobility of macromolecules in acrylamide gels (lo- 12), preliminary study may be advisable in most cases to determine gel compositions conferring both suitable mobility and satisfactory gel slicing properties. It would appear desirable to utilize high crosslinking and agarose concentrations to retain good slicing properties, and to adjust mobility by raising or lowering the concentration of acrylamide. Little skill was required for precise sectioning because the critical steps of gel advancing and slicing were, to a large extent, automated. The simplicity, rapidity, precision, and versatility recommend this device for most acrylamide gel slicing. ACKNOWLEDGMENT 1 am grateful for the skillful technical assistance of Ms. Drusilla Grant.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

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