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Gel Electrophoresis of the Major Coat Protein
Procedure of R.N. Perham, University of Cambridge
The type 88 vector f88-4 (see vectors.doc) produces virions with two types of major coat protein (pVIII) subunit: the 50-residue wild-type subunit and the recombinant subunit with the extra guest peptide. Assuming the phage have been propagated in 1 mM IPTG to fully induce the recombinant gene VIII, typically about 150 of the 3900 pVIII subunits (~4%) are recombinant. We estimate the proportion of recombinant protein crudely by separating recombinant and wild-type pVIII electrophoretically, and comparing the intensities of the wild-type and recombinant protein bands. For a somewhat more quantitative analysis, the weak recombinant band can be compared with the weak wild-type bands from a series of standards consisting of phage at1.25–10% of the concentration of phage in the sample lane. The gel system described below is able to resolve the 50-residue wild-type major coat protein (pVIII) from most recombinant pVIII’s that are even just a few amino acids longer. However, different recombinant proteins with the same length sometimes migrate very differently from each other. The volumes below are for a Hoefer SE400 apparatus with 1.5-mm spacers. We assume, too, that only fiveof the ten lanes are used, allowing the gel to be trimmed down and western-blotted on aminigel apparatus (we describe use of a Hoefer TE22 Mini Transphor minigel western-blotting apparatus below; obviously, other models are equally suitable; if more lanes are needed, use a large-gel western-blotting apparatus instead).
STOCK SOLUTIONS AND MATERIALS
Stock acrylamide: 49% acrylamide, 0.5% bis
Stock buffer: 3 M Tris.HCl pH 8.3
2 × sample buffer: 12 ml 10% SDS, 6 ml glycerol, 1 ml stock buffer, 11 ml water, 1.2 mg bromphenol blue
Towbin transfer buffer
1576 ml water
6.06 g Tris (25 mM)
28.8 g glycine (192 mM)
400 ml methanol (20% v/v)
Do not adjust pH (pH should be 8.1-8.4)
Store in refrigerator; can be re-used once
four 10 × 9 cm pieces of 3MM filter paper
one 10 × 8.5 cm piece of nitrocellulose filter paper
NOTE: Whenever handling the nitrocellulose, 3MM filter paper, or other components of the western blot “sandwich,” wear gloves. Avoid touching the nitrocellulose as much as possible, even with gloves.
Glass rod, about 10 cm long and well rounded at the ends
AP-SA (alkaline phosphatase-streptavidin conjugate) 0.5 mg/ml
We obtain this reagent from Jackson ImmunoResearch Laboratories (Tel. 800-367-5296; Code no. 016-050-084). It is supplied as 1 ml of a 1 mg/ml solution in 10 mM Tris.HCl, 250 mM NaCl, pH 8.0, with 30 mg/ml BSA. It is processed as follows for long term storage.
a. Centrifuge the vial briefly to drive solution to the bottom.
b. Add 1.26 grams (1 ml) of a premix consisting of 2.52 grams (2 ml) glycerol, 40 µl 1 M MgCl2, and 4 µl 1 M ZnCl2. Close the vial, and rotate it in the cold room 1 hr to ensure complete mixing.
c. Re-centrifuge the vial to drive the solution to the bottom. Aliquot the mixture into two 1.5-ml Ep tubes and store in the refrigerator.
The solution seems to last at least a year in these circumstances.
Pierce 1-step NBT/BCIP substrate solution
Biotinylated antibody against filamentous phage
Biotinylated polyclonal sheep antibody to M13 is available from 5´3´ (Tel. 800-533-5703; Catalogue no. 7-187156; $170 for 250 µl). We have made our own biotinylated rabbit antibody to fd; we don’t ordinarily distribute this, but we might be willing to on a case-by-case basis if someone convinces us that the commercial source doesn’t work.
POURING GEL
1. Make the resolving gel: In a 125-ml vacuum flask mix 9.42 grams (7.48 ml) glycerol, 5.9 ml water, 13.35 ml stock buffer, 13.25 ml stock acrylamide; degas; add 356 µl 25% (w/w) ammonium persulfate and 35.6 µl TEMED.
2. At the same time make the stacking gel: In another 125-ml vacuum flask mix 1.5 ml stock acrylamide, 3.75 ml stock buffer, 9.75 ml water, 150 µl 25% (w/w) ammonium persulfate, 10 µl TEMED.
3. Pour the resolving gel to a level about 1 cm below the bottom of the wells, then layer the stacking gel on it directly before the resolving polymerizes; rock gently to allow the layers to level out; insert a 10-well comb as usual. The lower resolving gel will polymerize before the stacking gel.
4. Make electrode buffer (for both buffer chambers): 0.1 M Tricine, 0.1 M Tris, 0.1% SDS.
PREPARING SAMPLES AND RUNNING GEL
5. Dilute phage sample in water to a final concentration of 4 × 1011 virions/ml. If you are trying to quantify the recombinant pVIII, you should also prepare a 2-fold dilution series with virions (canbe wild-type) at four concentrations ranging from 4 × 1010 to 5 × 109 virions/ml. In 1.5-ml Ep tubes mix 20 µl phage samples, 20 µl 2 × sample buffer and 2 µl mercaptoethanol (you can leave the mercaptoethanol out if it’s not necessary to break disulfide bonds; the wild-type pVIII has no cysteines).
6. Heat in a 90–100° water bath for a few minutes.
7. Load 25-µl samples (~5 x 109 physical particles, corresponding to~165 ng coat protein) in appropriate wells. Try to avoid the two outside wells; in fact, we usually use only five inner wells, since in that case we can trim the gel to a size that can be western-blotted in a minigel apparatus.
8. Run at up to 110 V until the bromphenol blue has run ~8.5 cm from the top of the resolving gel (typically overnight); this will put the wild-type pVIII band at ~6.8 cm.
NOTE: Proteins can can be detected chemically by stainingthe gel with a nickel staining kit (VWR Scientific #EK 117 4143; $192.50) as per instructions. However, this methodis not very good at detecting a low level of expression. In this case, it is probably better to western blot the gel and detect phage proteins (including pVIII)with anti-phage antibody as described below.
WESTERN BLOTTING
NOTE: Wear gloves whenever you handle the gel or the other components of the western blot sandwich, particularly the nitrocellulose.
9. Trim the stacking gel off the resolving gel. Trim the resolving gel to ~9 cm wide (should easily accommodate 5 lanes) and 8.3 cm long (just above the bromphenol blue band).
10. Rock the gel in a large plastic box in Towbin transfer buffer for ~60 min, changing the buffer at least once.
11. Soak the two minigel fiber pads, the four 3MM sheets and the nitrocellulose filter in Towbin transfer buffer. Before wetting the nitrocellulose, mark the upper left-hand edge "TOP" with a soft pencil; this will be the face of the filter that faces the gel during the Western blot transfer; we’ll call this face the “antigen face.” Wet the nitrocellulose as usual by laying it on the buffer and allowing the water to gradually permeate the membrane before submerging the membrane.
12. Put a small piece of red tape on one of the two panels of the western-blot “sandwich”; this will be the panel that faces the positive electrode during electrophoretic transfer. Fill a plastic dish (large enough to accommodate the western blot “sandwich”) about half full of Towbin transfer buffer. Put the panel without the red tape (the one that will ultimately face the negative electrode) flat on the bottom and the clear panel (with the white plastic latch) vertical.
13. Fill the buffer tank about 3/4 full of Towbin transfer buffer.
14. Place one of the pre-soaked fiber pads on the bottom panel, expelling any air by rolling with the glass rod like a rolling pin. Lay one of the pre-soaked 3MM sheets on top of the fiber pad, being careful to center it.
15. Pour off the Towbin transfer buffer from the dish with the pre-soaked gel, making sure the gel lies flat on the flat part of the floor of the dish; lay one of the pre-soaked sheets of 3MM onto the gel, and roll gently with the glass rod to lightly “glue” the 3MM to the gel. Holding the 3MM filter paper, turn the whole assembly upside down, and peel or tap the gel off the dish and onto the 3MM filter paper. Lay the 3MM, gel side up, onto the other 3MM filter paper on fiber pad on the panel.
16. Overlay the gel with the pre-soaked nitrocellulose filter, antigen face down (toward the gel) and with “TOP” at the top of the gel, and roll out any bubbles with the glass rod.
17. Overlay the nitrocellulose with the second then the third pre-soaked sheets of 3MM, again expelling bubbles with the glass rod.
18. Place the second pre-soaked fiber pad on top of the 3MM, rolling out bubbles with the glass rod.
19. Lay the panel labeled with red tape (the one that will face the positive electrode) on top of the second fiber pad. Check that nothing overhangs the edges of the cassette, particularly the side edges. Working as fast as possible, pinch the panels of the cassette together,lift the “sandwich” out of the dish, and slide it into a slot in the buffer chamber, the panel with the red tape facing the positive electrode. Add additional Towbin transfer buffer as necessary to bring level just over the upper edge of the cassette.
20. Connect power supply to the electrodes as usual (red positive, black/smoky negative) and run overnight at 20V. At this low voltage, the buffer doesn’t overheat.
21. Disconnect the power supply; remove the sandwich and lay it on a bench with the panel labeled with red tape uppermost. Carefully peel off the upper fiber pad and 3MM paper, exposing the nitrocellulose still clinging to the gel. Using a 26-gauge needle, outline the contour of the gel with pin-pricks in the nitrocellulose. Then carefully peel off the nitrocellulose (if any pieces of gel adhere to it, you must remove them by gently rubbing with your gloved finger under the tap bofore proceeding) and lay it antigen side up in a plastic box.
STAINING THE WESTERN BLOT WITH ANTI-PHAGE ANTIBODY
22. To the box with the blot add ~100 ml 5% non-fat dry milk dissolved in TBS (0.15 M NaCl, 50 mM Tris.HCl pH 7.5). Allow the blot to block at room temperature for 1 hr on the rocker.
23. Rinse the blot with TBS/Tween (TBS with 0.5% v/v Tween 20) several times.
24. Rock for 2–3 hr in 10–20 ml TTDBA (TBS/Tween supplemented with 1 mg/ml dialyzed BSA (Sigma) and 0.02% NaN3) containing 10 µg/ml biotinylated anti-phage antibody (other antibodies might have to be used at different concentrations).
25. Wash the blot several times in TBS/Tween.
26. In a large weigh boat (if possible; otherwise, use a plastic box) measure 10 ml TBS/Tween,200 µl 50-mg/ml dialyzed BSA and mix by tipping; then add 40 ml 0.5-mg/ml AP-SA and mix by tipping box. Using forceps to lift blot by corner, give the blot one final rinse with TBS/Tweenand put it into the diluted AP-SA. Incubate on rocker 10 min at room temperature.
27. Using a forceps, lift the blot by a corner, rinse it with TBS/Tween from a squirt bottle, and tranfer it to a large weigh boat containing ~30 ml TBS/Tween. Rock for a few min, then lift the blot with forceps, rinse it with TBS/Tween from a squirt bottle, transfer it to another weigh boat containing ~30 ml TBS/Tween, and rock it ~10 min at room temperature. Wash blot twice more in ~30 mlTBS/Tween ~5 min. Put the blot in clean weigh boat.
28. Mix the Pierce 1-step NBT/BCIP substrate solution well and pour into weigh boat to just cover blot; allow to rock at room temperature ~5–15 minutes until color develops in the sample lane.
29. Terminate development by washing with water and then allowing to stand in 10×TE. Air dry. You can store the blot itself, or you can photograph it (preferably through a yellow filter).
Assuming you’ve used a polyclonal antipphage antibody, you’ll see a prominent pIII protein band near the top of the blot; though the mass of pIIImolecules amounts to only ~1% of the mass of pVIII molecules, pIII an immunodominant antigen. The wild-type pVIII band will be a prominent band toward the bottom of the gel (~80% of the migration of bromphenol blue). The recombinant pVIII band, if present, will lie above, and be much fainter than, the wild-type pVIII band. If you’ve run dilution standards with graded intensities of the major pVIII band, you should be able to quantify the minor recombinant pVIII band in the sample.