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Fig. 4. Preparative ACE schematic. Top panel: A preparative ACE gel is poured using a casting stand as shown in Fig. 5, except instead of using protein well-forming Teflon combs, a single Plexiglas block is used to create one large rectangular well to be filled with a single protein-agarose mixture. Radiolabeled GAG or PG is loaded into the slot above the protein-containing wells (shown as a dark line to the left in the gel schematic), and electrophoresed through the protein-containing zone. Middle panel: The agarose gel surrounding the protein-containing zone is trimmed away, and the remaining protein-agarose block is sectioned into 2-mm-thick segments. The amount of radiolabeled GAG or PG in each segment is then determined. Bottom panel: Actual plot of CPM/fraction of heparin octasaccharide mixture electrophoresed through 1000 nM type I collagen, showing the partial resolution of four differentially binding populations. (From San Antonio and Lander, unpublished data). Artwork by Drew Likens.

which undergo fibrillogenesis within about 20 min after being brought from an acidic to a neutral solution, such fibrils are insoluble and are impossible to subject to a serial dilution, as is required in ACE. Thus, to avoid this problem one must bring collagen solutions from the acid soluble to the neutralized state, and then mixed into agarose and pipetted into ACE gels before fibrillogenesis occurs (4,5).

Fig. 5. Oblique view of apparatus for pouring two ACE gels, each with protein-containing lanes 15 mm in length. Plexiglas casting stand contains a clear piece of gel bond (not visible in this photograph), on which are placed two Teflon combs that are each used to create 9 agarose-protein-containing lanes, and two Teflon strips that are each used to create a GAG/PG loading slot. The stand is bordered on two sides by masking tape, which retains the agarose and Teflon strips in place. After filling the stand with agarose, upon solidification the combs and strips are removed, forming two ACE gel templates to be run as described in the text and shown diagramatically in Fig. 1.

Fig. 5. Oblique view of apparatus for pouring two ACE gels, each with protein-containing lanes 15 mm in length. Plexiglas casting stand contains a clear piece of gel bond (not visible in this photograph), on which are placed two Teflon combs that are each used to create 9 agarose-protein-containing lanes, and two Teflon strips that are each used to create a GAG/PG loading slot. The stand is bordered on two sides by masking tape, which retains the agarose and Teflon strips in place. After filling the stand with agarose, upon solidification the combs and strips are removed, forming two ACE gel templates to be run as described in the text and shown diagramatically in Fig. 1.

1.3. Are the GAGs or PGs of Interest Suitable?

One of the requirements of ACE is that the GAG or PG concentration is much less than the Kd of GAG- or PG-protein binding (1). Thus, radiolabeled GAGs and PGs are used at trace concentrations in ACE gels, i.e., generally far less than 1 ^g of GAG or PG/gel will suffice. However, the GAG/PG must be radiolabeled or labeled otherwise, and present in great enough quantities to be detected (generally for radiolabeled samples at least 10,000 cpm/gel). Therefore, the GAGs or PGs must be metabolically radiolabeled with 35S-sulfate or 14C-D-glucosamine in culture and subsequently purified, or purified in their native forms but derivatized with, for example, Bolton-Hunter reagent (6), fluoresceinamine (1), or tyramine (3), followed by radioiodination. Here we have presented a method for the tyramine endlabeling of heparin for use in ACE. Another factor that must be considered in the analysis of data from ACE gels is the potential multivalency of GAGs or PGs in terms of their interactions with proteins; this issue is addressed elsewhere (1).

2. Materials

1. ACE casting apparatus (see Fig. 5): casting stage(s), protein well-forming comb(s), PG/GAG lane-forming comb(s), and tape (autoclave or equivalent). Combs are precision tooled from Teflon blocks (for protein well-forming combs) or sheets (for PG/GAG lane-forming combs). The ACE casting apparatus we most commonly use includes a casting stage made of Plexiglas with a gel platform of 100 long x 75 wide x 6 mm deep; protein well-forming combs consisting of nine parallel rectangular blocks spaced 3 mm apart, each 15 long x 4 x 4 mm; and PG/GAG lane-forming combs cut from a 25 x 75 mm rectangle of 1-mm-thick Teflon. Rectangles of 4.5 x 10 mm are removed from two corners to produce a comb with one 66-mm edge, which is stood on its short edge and held upright in the casting apparatus by pressing the tape used to seal the apparatus against the overhanging tabs of the comb.

2. At least 1.2 L of running buffer (RB). To make 2 L, add 22.53 g of sodium 3-(N-mor-pholino)-2hydroxypropanesulfaonate (MOPSO) to 1.8 L of distilled water with stirring. Add 20.51 g of sodium acetate, anhydrous, or 34.02 g of sodium acetate, trihydrate. Use 5 M NaOH to bring the pH to 7.0. Bring to 2 L with distilled water. Store at 4°C, will last no longer than several months. This buffer can also be prepared as a 5x concentrated stock and diluted before use.

4. 1.053% agarose: 1 g of low-melting-point (LMP) (Sea Plaque Agarose; FMC) in 95 mL of RB.

5. 2.22% agarose: 1 g of LMP agarose (Sea Plaque Agarose; FMC) in 45 mL of RB.

6. 10% CHAPS in distilled water.

7. Leveling device.

8. ACE gel running box (we use the Hoefer Super-Sub apparatus).

9. Low voltage electrophoresis power supply, capable of delivering 75 V.

10. Waterbath set to 37°C.

11. Boiling-water bath or microwave oven.

12. Circulating water chiller (unnecessary if cold-water tap is available at lab bench where the gel will be run).

13. Small space heater (1500 W).

3. Methods

3.1. Analytical ACE

This protocol is used to estimate the Kd of GAG or PG binding to a protein, or to visualize heterogeneity in binding between a PG or GAG mixture and a protein.

1. Place the casting stand on a level benchtop. On a piece of GelBond, determine which is the bonding side by placing a drop of distilled water on one of the sides. If the drop beads up, it is the nonbonding side and is placed down on the casting apparatus; if the bead spreads out, it is the bonding side and is placed face up. Using scissors, cut the GelBond to fit the casting apparatus, then use 1-2 drops of distilled water to hold the nonbonding side in place. Press excess water out from between the GelBond and the casting stage and, using Kimwipes, make sure that the sides of the stage are dry.

2. Use autoclave tape to seal the edges of the casting stage, making sure to leave about 1 in (2.5 cm) excess on both ends so it can be folded against itself to make a tab to facilitate its removal later. Place the sample-forming comb(s) in the casting stand using the tape to hold it in place, then place the protein-lane forming comb by centering it and leaving a space of about 2 mm between it and the sample comb. Using the 15-mm-long protein well-forming combs and the gel casting stand specified here, either one or two ACE gels can be poured per stand.

3. When agarose is made fresh, to promote its rapid dissolution allow at least 20 min for it to soak in room temperature RB before the mixture is boiled. Place a glass bottle containing the 1.053% agarose into the water bath, making sure it cannot tip and that the cap is very loose, so that upon heating it will not explode. The bottle should be left in the boiling-water bath or microwaved until the agarose has come to a boil and is completely melted. Check that the agarose is completely mixed by swirling the bottle to see that the liquid appears homogeneous.

4. To a polypropylene tube, add 1.0 mL of 10% CHAPS and bring to volume with 20 mL with 1.053% boiling hot agarose, place the lid on tightly and gently invert a few times; take care not to create bubbles. Pour the agarose quickly into the casting apparatus, making sure that the agarose flows evenly between the combs. If any of the combs shifted in position during the pouring, readjust them into proper position while the agarose is still hot. Allow the agarose to solidify completely before removing the tape (usually at least 30 min). If any agarose leaks out of the casting stand, or there are significant numbers of bubbles remaining in the agarose after it has solidified, the gel must be discarded.

5. While the gel is cooling, perform a serial dilution of your test protein, into nine concentrations including several that should exceed the Kd, several that are close to it, and several that should be considerably lower (see Note 1). Number nine polypropylene tubes and add the correct amount of diluent, usually RB, to each. Usually, each sample is diluted in RB to twice its desired final concentration, since later they will be mixed 1/1 with 2.0% agarose before being loaded into ACE gels (see step 8). An exception is for collagen samples, which are dissolved in 0.5 N acetic acid at eight times their final concentration, and are quickly mixed 1/1 with 0.5 N NaOH, and then 1/1 with 2x RB, before being mixed 1/1 with 2.0% agarose.

6. After the gel has solidified, remove the well-forming combs first, by slowly and gently sliding the comb backwards until it separates from the agarose at the front end, then sliding it forward until it separates from the agarose at the rear end. Then, while holding the casting stand firmly against the bench surface, tilt the comb and gently pull it away from the agarose. Be careful not to rip the gel or deform the lanes. Small rips can be repaired by reinserting the comb and pipetting hot 1.0% agarose (prepared as before) into the gel. Should small clumps of agarose remain in sample lanes, these can be removed using a pipet tip affixed to a vacuum line. The PG/GAG lane-forming comb is next removed by holding the comb in place while slowly peeling the tape away from each of its sides, then the comb is lifted straight up, out of the agarose. If rips occur in the agarose between the sample-forming slot and the gel edge, these can be repaired by reinserting the comb and pipetting new 1.0% agarose (prepared as before) onto the damaged regions of the gel. If the PG/GAG lane-forming comb has been removed before the gel is fully solidified, the sample well may collapse upon itself and the gel must be discarded. Remove the gel (which should be firmly attached to the gel bond) from the casting stand. Using a waterproof marker, mark on the gel bond the position of the PG/GAG loading lane, and any necessary notes about the samples to be added to the protein-containing wells.

7. To a polypropylene tube, add 10% of the total volume needed of 10% CHAPS and bring to volume with boiling-hot 2% agarose, place the lid on tightly and gently invert a few times to mix. Using the ACE gel dimensions specified under Subheading 2., each ACE gel will require 1.125 mL of CHAPS/2% agarose, although extra volume should be prepared, as some is lost on the side of the sample tube and on the outside of pipet tips. Place the tube in the 37°C bath to equilibrate for later use (see Note 2).

8. To load the test proteins into the gel wells, make sure the gel is level and located near the 37°C water bath. From the 2% agarose tube, withdraw an amount equal to the total volume within each of the nine sample tubes, which would be 125 ^L. Add this volume to the ninth sample tube (which contains the most dilute protein sample) and mix it by agitating the pipet tip back and forth while rapidly drawing the solution in and out of the tip 10 times, taking care not to generate bubbles in the mixture. Then add the mixture (now totaling 250 ^L) to lane 9, which is the rightmost of the nine lanes of the gel, taking care to overfill the lane slightly. Any excess agarose that spills over into the zone between sample lanes will quickly solidify and will not interfere with the running of the gel, whereas underfilling the protein lanes results in anomalous GAG/PG migration through the gel. Move on to the remaining samples, filling the lanes in the following order: 7, 5, 3, 1, 8, 6, 4, and 2. The key to success during this step is to mix the samples thoroughly but to make sure to work quickly enough that the agarose-protein mixture does not gel in the tube. If need be, the mixing can be done while the sample tube remains partially submerged in the water bath.

9. Prepare the PG or GAG sample by mixing the labeled material in sufficient quantity and activity that it can be detected (usually at least 10,000-20,000 cpm of 35S-radiolabeled material is sufficient for a good 3-d exposure in a phosphorimager cassette); tracking dye(s) (we use 0.05% each of bromophenol blue and xylene cyanol); sucrose so that the sample will sink through the RB during gel loading (5% w/v), and enough RB to bring the volume to approximately 100 ^L/gel (see Note 3). Once the labeled sample is mixed, it should be vortexed and any insolubles pelleted at 13,000g for 2 min. When adding the sample to the gel, be careful not to disturb any pellet that may be at the bottom of the tube.

10. The electrophoresis apparatus is next prepared. Make sure the apparatus is resting properly on a level stir plate. Attach the cooling hoses to a water source, either cold tap water or a circulating water chiller set to 10°C, and turn on the water. Fill the apparatus with cold running buffer to roughly 3 mm above the platform, then place the ACE gels with the PG/GAG lane end of the gel oriented closest to the cathode, and use glass microscope slides to hold them in place. Turn on the stir plate to purge air trapped in the buffer recirculator, then turn the stir plate off. Add more buffer if needed, to be sure that the gels are covered by about 3 mm of buffer. Using a pipetter with a 200-^L gel-loading pipet tip, load the labeled sample into the lane, drawing it across the gel while discharging the sample, so that it fills evenly. Take care not to nick the gel with the pipet tip during sample loading—this leads to an uneven sample front and/or rapid loss of the sample into the running buffer during electrophoresis (see Note 4).

11. Attach the cover to the gel box, plug the leads into the power supply, and turn it on to deliver 60-80 V. After the tracking dye has entered the gel (in approximately 5 min), turn on the stir plate to an intermediate setting but take care that the resulting agitation of the buffer does not cause the gels to float away from the platform.

12. Run the gel for the appropriate time. For example, for gels containing protein-containing lanes 15-mm-long, heparin samples should generally run for about 1.0 h, by this time the heparin should have migrated most if not all of the way through the lane. PGs and other GAGs may migrate at different rates depending on their relative sizes, charges, etc. The position of the tracking dyes can be used as a guide to assess when the gels are finished— for example, the dyes that we use (see step 9) typically move at approximately one-third the rate of heparin in the electrophoretic field. After electrophoresis, turn off the power supply and remove the gel box cover. Remove the gels and place them on an elevated surface that will not impede the flow of warm air around them. We use 8-cm-high plastic test tube racks placed about 30 cm in front of a warm air source supplied by a small personal space heater, such as the Holmes (HFH 195, 1500 W). Allow the gels to dry at the high heat setting for at least 8.0 h; they are dry when the agarose has flattened to a thin clear sheet that is not sticky to the touch.

3.2. Data Analysis

ACE gel electrophoretograms can be visualized by autoradiography or phosphorimaging, and the approximate Kd of GAG- or PG-protein binding can often be estimated by visual inspection. For example, from the phosphorimages of ACE gels shown in Fig. 2A, it can be seen that the affinity of syndecan type I collagen binding can be estimated as 50 nM < Kd < 250 nM, since within these concentration ranges it is evident that the PG is half-shifted from being fully retarded at high protein concentrations to being fully mobile at very low protein concentrations. However, such gels can also be analyzed quantitatively, by first measuring GAG or PG mobility using a Phosphorimager by scanning protein-containing lanes and determining relative radioactivity per 88-^m pixel along the length of each lane (see Fig. 2B) (3). GAG or PG mobility is taken as the pixel position that divides the curves representing the distribution of GAG or PG into halves of equal areas. The retardation coefficient R is next calculated for each lane as the GAG or PG migration position in that lane divided by its mobility in a protein-free lane (R = (Mo-M)/Mo, where Mo is the mobility of free GAG or PG, and M is its mobility through protein; see Fig. 1). Under appropriate experimental conditions as described previously, R is proportional to the fractional saturation of GAG or PG by protein, so that values of the equilibrium binding constant may be determined from the relationship between R and protein concentration (1). Data are analyzed graphically, by curve-fitting to the equation R = RJ(\ + Kd/[protein]n; where Rx represents the value of R at full saturation (i.e., at an arbitrarily high protein concentration), and n is a coefficient that reflects cooperativity of binding (1). Nonlinear least-squares fits are calculated using a graphics program, e.g., the Kaleidagraph program (Synergy Software, Reading, PA). The above analysis provides apparent Kd values for GAG- or PG-protein interactions, but does not indicate whether the sample of GAG or PG exhibits heterogeneity in protein binding. Such heterogeneity is usually apparent by visual inspection of the ACE gel electrophoretogram, as evidenced by a broad smearing of the GAG or PG migration front throughout the length of protein-containing lanes, or by the presence of multiple bands of the sample at concentrations near the binding Kd (see Fig. 3). When binding heterogeneity is evident, subpopulations of GAGs or PGs that bind strongly or weakly to protein can be isolated using preparative ACE, and subjected to further analysis.

3.3. Preparative ACE

This method is used to fractionate a heterogeneous population of GAGs or PGs that bind differentially to a protein. In this technique, radiolabeled GAGs or PGs are subjected to electrophoresis through agarose containing a single concentration of protein (1,3) as shown schematically in Fig 4. After individual species from the GAG or PG mixture are isolated, their affinity for protein can be analyzed by standard ACE methods, or they can be subjected to other analyses.

1. A 1% low-melting agarose/CHAPs solution in ACE electrophoresis buffer is prepared as detailed previously and is poured hot onto a piece of GelBond fitted within a Plexiglas gel casting tray where a 4 x 7 cm Plexiglas block and a Teflon PG/GAG lane-forming comb are positioned. After the agarose solidifies, removal of the block and strip leaves a 4 x 7 cm well with a 66 x 1 mm slot 2 mm away from and parallel to one of the short edges of the 4 x 7 cm well (see Fig. 4, top).

2. A protein is prepared at a concentration that will achieve maximal separation of GAG or PG species of interest as determined by analytical ACE (e.g., for the protein in Fig. 3, a concentration of 250 nM may be suitable). The sample is then mixed with agarose as described in step 8 of the analytical ACE methods to a final agarose concentration of 1.0%, and is loaded into the 4 x 7 cm well and allowed to solidify.

3. Gels are submerged under electrophoresis buffer, radiolabeled GAG or PG is loaded into the 66 x 1 mm slot, and electrophoresis is carried out, all as detailed in steps 9-12 of the analytical ACE methods.

4. After electrophoresis, gels are removed and are not dried, but rather are affixed to a surface marked with a millimeter grid, with the electrophoretic origin at the top. For a surface we use a sheet of 1-mm lined graph paper under a clear plastic sheet, taped onto a flat Styrofoam block.

5. Regions of the gel to the left and right of the 4 x 7 cm block as well as the 3 mm of the block itself that are immediately adjacent to the left and right edges are cut away and discarded.

6. The resultant 3.4 cm x 7 cm gel is sectioned into 2-mm segments perpendicular to the direction of electrophoresis, using a piece of surgical suture thread drawn tightly between the hands.

7. Each agarose segment is lifted with a flat-headed metal spatula and placed in a tube, and the amount of radiolabeled GAG or PG in each segment is measured with a gamma counter, or by liquid scintillation counting of a melted gel aliquot. To melt gel segments, tubes are placed in a >70°C water bath. They may be pooled or divided into aliquots while they are liquid, and then stored frozen. Alternatively, they may be melted at 70°C and then brought to 6 M in urea by addition of solid urea (urea blocks gelation of the agarose). In this case they will remain liquid for days, even at 4°C. Such samples may then be mixed with running dyes and loaded directly into analytical ACE gels (see Note 5), or subjected to other analyses.

3.4. Iodination and Molecular-Weight Fractionation of Heparin

Heparin is often used as a model compound in studies of GAG- or PG-protein interactions because it potentially contains a large number of different protein-interactive domains, is structurally homologous to heparan sulfates that are common to many cell surface and extracellular matrix PGs, and is inexpensive and readily available from commercial sources. Commercial heparin is polydisperse in Mr, often averaging about 15 kDa. However, in protein-binding studies the use of low-molecular-weight heparin is advantageous, since it minimizes factors that complicate binding analysis, such as multivalency (1). Thus, here we have included methods we use to tyramine-derivatize, radioiodinate, and Mr-fractionate commercial heparin to be used in ACE analysis. We have found that derivatization of heparin with tyramine is more suitable than with fluoresceinamine, as the latter may artifactually enhance heparin-binding affinity for protein (3).

3.5. Tyramine Labeling of Heparin

1. Dissolve 9 mg of heparin in 750 ^l of 5% (w/v) solution of tyramine in formamide, and place in a sealed tube. We use porcine intestinal mucosa heparin (grade 1A; Sigma) in our experiments.

2. Heat to 80°C for 1 h and cool to room temperature (the solution may turn yellow).

3. Add 1 mg of Na cyanoborohydride, seal, and incubate overnight at room temperature.

4. Dilute with 9 volumes of distilled water.

5. Dialyze against distilled water using 1-kDa Mr cutoff dialysis tubing.

6. Lyophilize the sample.

7. Resuspend in a small volume of water and measure heparin concentration [e.g., using the Dische assay (7)].

8. Measure tyramine content by OD278. As heparin absorbs to a small extent at 278 nm, tyramine concentrations must be corrected by subtracting this background. Tyramine content may be estimated from corrected OD values by the formula: tyramine (mg/mL) = 0.0824 x OD278.

3.6. lodination of Tyramine-Heparin

1. Dissolve 400 ^g of Iodogen/mL (Pierce) of dichloromethane. Add 50 ^L to the bottom of 5-mL glass test tubes; a glass Pasteur pipet connected to a rubber hose affixed to a nitrogen tank can be used to direct a slow stream of nitrogen gas over the sample in the bottom of the rotating tube. Tyramine-coated glass tubes can be covered with Parafilm and stored under vacuum at room temperature for years.

2. Dilute 2.5 ^g of tyramine-labeled heparin in 50 ^L of 0.25 M Tris-HCl, pH 7.5.

3. Rinse an Iodogen-coated tube gently with 500 ^L of 0.25 M Tris-HCl, pH 7.5 to wash off any unbound Iodogen. Visually inspect tube to ensure that the Iodogen coat remains intact.

4. React 50 ^L of heparin solution and 5 mCi of 125I at room temperature for 6 min with intermittent agitation, then add an additional 50 ^L of buffer for 6 min more with agitation. The addition of extra buffer helps prevent the Iodogen from increasing the pH, which inhibits iodination.

3.7. Desalting of125-Heparin on G-25

After iodination of the heparin sample, it must be desalted over a G-25 column (prepared in a 2.0-mL disposable tissue culture pipet) to remove unbound 125I.

1. Equilibrate and elute the column with 0.5 x RB.

2. Draw off buffer from column bed.

3. Load the 125I-heparin sample onto the column and position a test tube rack with numbered Eppendorf tubes underneath.

4. Collect the column eluate in fractions of 2 drops/tube.

5. Make sure to replenish RB as the column runs.

6. Monitor the passage of 125I-heparin through the column using a Geiger counter.

7. As fractions are collected, monitor their relative radioactivity using a Geiger counter at a fixed distance from tubes.

8. Record activities and plot elution profile to determine where bound (the smaller of the two peaks, which elutes first) and free (the larger of the two peaks, which elutes second) isotope are eluting. Generally, 10-15 fractions are collected.

9. Pool the fractions containing the bound isotope.

10. Discard the column and the fractions containing the unincorporated isotope.

3.8. Molecular-Weight Fractionation of125-Heparin on G-100

Pooled fractions containing 125I-heparin from the desalting column are prepared for G-100 chromatography and Mr-fractionated as follows; our typical column dimesions are 300 x 10 mm. A typical elution profile of 125I-heparin from a G-100 column is shown in Fig. 6.

1. Prepare the sample for G-100 chromatography by mixing 125I-heparin (generally < 0.5 mL) to 5% sucrose (w/v) plus 10 ^L of a saturated phenol red solution. Bring to 2.0 mL with running buffer, clarify at 13,000g for 2 min, and load on column.

2. Collect fractions of about 8-10 drops (i.e., from 0.5-1.0 mL).

Fig. 6. Elution profile of 125I-tyramine-heparin from G-100 column. The larger of the two peaks, eluting first, represents the radiolabeled heparin sample; the smaller peak, eluting second, is unincorporated 125I-iodine. The last 12% (marked with arrows) of sample to elute represents the low-Mr heparin chains of about < 6 kDa.

Fig. 6. Elution profile of 125I-tyramine-heparin from G-100 column. The larger of the two peaks, eluting first, represents the radiolabeled heparin sample; the smaller peak, eluting second, is unincorporated 125I-iodine. The last 12% (marked with arrows) of sample to elute represents the low-Mr heparin chains of about < 6 kDa.

3. Remove and count several microliters of each fraction using a gamma counter.

4. Plot column profile indicating elution position of phenol red, which should largely overlap with free iodine peak. See Fig. 6 for an example of a typical profile.

5. The first 12% of radioactive material to elute is the high-molecular-weight fraction.

6. The following 76% to elute is the medium-molecular-weight fraction.

7. The remaining 12% to elute is the low-molecular-weight fraction of Mr <= 6 kDa (8-10).

3.8.1. Storage of125-Heparin Samples

1. Pool fractions within each category and cryoprotect with bovine serum albumin (BSA) to 0.1 mg/mL (a 2.0-mg/mL stock of BSA can be used).

2. The low-Mr fraction, which is the fraction most commonly used in binding experiments, should be divided into 50- to 100-^L aliquots.

3. The remaining fractions should be divided into samples of about 1.0 mL.

4. Store samples at -80°C. These can be used for approximately 3-6 mo.

4. Notes

1. The accuracy of the ACE technique relies on knowing the exact concentration of the test protein.

2. If the 2% agarose mixture is not equilibrated to 37 °C when it is mixed with test proteins (see step 8 under Subheading 3.1.), the proteins may become denatured.

3. Tubes containing 5x stock mixtures of dyes/sucrose should be premixed, filtered, and stored frozen.

4. Loading of the GAG/PG sample is the most technically difficult step of this procedure; thus, one should practice loading mock samples into ACE gels before working with radioactive GAG or PG samples.

5. Since urea does not migrate in the electrophoretic field, its removal from samples before electrophoresis is not required; due to the density of urea, it is not necessary to add sucrose.

References

1. Lee, M. K., and Lander, A. D. (1991). Analysis of affinity and structural selectivity in the binding of proteins to glycosaminoglycans: development of a sensitive electrophoretic approach. Proc. Natl. Acad. Sci. (USA) 88, 2768-2772.

2. Lim, W. A., Sauer, R. T., and Lander, A. D. (1991). Analysis of DNA-protein interactions by affinity coelectrophoresis. Meth. Enzymol. 208, 196-210.

3. San Antonio, J. D., Slover, J. Lawler, J., Karnovsky, M. J., and Lander, A. D. (1993). Specificity in the interactions of extracellular matrix proteins with subpopulations of the glycosaminoglycan heparin. Biochemistry 32, 4746-4755.

4. San Antonio, J. D., Lander, A. D., Wright, T. C., and Karnovsky, M. J. (1992). Heparin inhibits the attachment and growth of Balb c/3T3 fibroblasts on collagen substrata. J. Cell. Physiol. 150, 8-16.

5. San Antonio, J. D., Karnovsky, M. J., Gay, S., Sanderson, R. D., and Lander, A. D. (1994). Interactions of syndecan-1 and heparin with human collagens. Glycobiology 4, 327-332.

6. LeBaron, R. G., Hook, A., Esko, J. D., Gay, S. and Hook, M. (1989). Binding of heparan sulfate to type V collagen. J. Biol. Chem. 264, 7950-7956.

7. Dische, Z. (1947). A new specific color reaction of hexuronic acids. J. Biol. Chem. 167, 189-192.

8. Yamada, K. M., Kennedy, D. W., Kimata, K., and Pratt, R. M. (1980). Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J. Biol. Chem. 255, 6055-6063.

9. Laurent, T. C., Tengblad, A., Thunberg, L., Hook, M., and Lindhal, U. (1978). The molecular-weight dependence of the anti-coagulant activity of heparin. Biochem. J. 175, 691-701.

10. Jordon, R., Beeler, D., and Rosenberg, R. D. (1979). Fractionation of low molecular weight heparin species and their interactions with antithrombin. J. Biol. Chem. 254, 2902-2913.

11. Verrecchio, A., Germann, M. W., Schick, B. P., Kung, B., Twardowski, T., and San Antonio, J. D. (2000). Design of peptides with high affinities for heparin and endothelial cell proteoglycans J. Biol. Chem. 275, 7701-7707.

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