Oliver Smithies:
[00:00:00] So this is Book i, beginning on August 22nd, 1974, and ending in October of the same year. We’re pursuing another topic which was eventually worked out completely with Byron Ballou, which is the use of gels with chloral hydrate, which was his favorite compound for dissolving anything, including plastic apparatus, as we found out you couldn’t use Lucite, you had to use polycarbonate — I’d forgotten what it is, not acrylic — anyway, you couldn’t use [00:01:00] the ordinary Lucite gel. You had to use a polycarbonate-type of gel.So on Thursday, August 22nd, page 1, the beginning of the work with slab gel with chloral hydrate, with buffer that contained formic acid, formate, pH 3.7, photopolymerized successfully. Realized the slots were too narrow, and the gel came away from the polycarbonate, etc., etc., on page 3. So that was a polycarbonate [00:02:00] apparatus. One, we tried last, and it leaked, didn’t polymerize.
Continuing with that type of experiment on Friday, August 23rd, trying to get the gel to work. And, a comment which is almost certainly written by Byron because of the language, “Use same procedure as August 22nd, except it was degassed thrice.” There’s nobody else that I know who would describe it as “thrice.” [00:03:00]
Test a gel on page 7, ran for 30-45 minutes, maximum voltage, and fairly straight at about 2cm. Stained with amido black. Anyone can see the sample which was a hemoglobin, rather faint. [00:04:00] Talking about making up the solutions, one can make up the polyacrylamide beforehand and store; you do not need to degas, page 7.
And a glass back apparatus was tried on Monday, August 26th, with a reasonably satisfactory migrations at a beginning stage, using Ed Sheldon’s Friday, August 23rd solutions, with a glycine chaser, [00:05:00] and it is not clear what the solutions of protein were, that were put on the gel.
Try a continuous gel on page 13, because the other was a discontinuous gel, discontinuous buffer system with a glycine chaser, and this was a continuous gel, now with a simple system of chloral hydrate glycine buffer, and so on.
A rather unsatisfactory result on the following page, 14. Approximately [00:06:00] 0.1M glycine continuous, but a pretty awful gel. Higher ionic strength tested on page 17, with a very poor result on the 0.05M formic continuous gel, but another less than 0.05M formic gel, which was quite reasonable. That’s approximately 0.02M, not accurate composition, it says. Various troubles documented. Going to 0.01M formic continuous, poor [00:07:00] result, and nothing very hopeful yet, and very good slots on page 23, but a pretty poor gel. Still bad. So retry ionic strength, higher ionic strength. Not much better. High ionic strength, and Byron suggests adding amino-methane-sulfonic, but the gel was pretty awful.
Continuing, retry Byron’s standard gel, commented on, but the gel on page 27 was 0.01M formic with a glycine chaser. [00:08:00] And 0.05M with a glycine chaser, on page 29. And, on page 29, there’s a comment, “Not a good test. Remember aluminum lactate,” where that memory came from, I know that we had used aluminum lactate before in doing the histones. So 0.04M aluminum lactate, which was [00:09:00] tried, I think, with hemoglobin. It’s not — it never said, but the comment that even [heme?] stayed on, suggests that it was indeed hemoglobin. “Very sticky gel, increase the bis. Looks very good. Best looking heme yet.” Etc., so the aluminum lactate gel is coming back into fashion.
And the result the next day is looking very good. “20V/cm, but only 3mA current with 0.04M aluminum lactate pH 2.3, [00:10:00] test beef heart mitochondria tomorrow,” etc. “Heme seems sharper on the aqueous solution visually. Try increasing the bis; the gel is rather gel-like.” Continue with different tests.
Here is a test of autoradiography, beginning on page 37, taking the medium with about 100,000 I-125 counts per minute, and clear also [00:11:00], this is 125-iodinated beta-2 microglobulin. It is bona fide and the medium from H-137 was used, precipitated with trichloroacetic acid, washed with acetone, and was fully dissolved very easily in the chloral hydrate, aluminum lactate.
The gel stained on Monday, about 130 counts in 5mL [00:12:00] of the gel, “Nice so need not throw out always, or at least don’t have to take great care.” But then one of these complex test film racks with top the gel, and [Saran?], and the film, etc., and developed 10AM on Monday, presumably people set up on Friday, they were not very clear. But the films are there, and there is radioactivity at the front that probably, three counts, from the look of it. Let’s see what it says. [00:13:00] Gel was stained on Monday, about noon. And then, an image of the gel after staining, very complicated pattern of proteins. And the test film was developed about 11AM on Sunday, beta-2 microglobulin in sample A was visible near the end of the gel, and exposed again until Tuesday. So here, on Tuesday, it’s very — retest of the antigen, retest of the [00:14:00] 125-iodinated sample with staining on Saturday, August 31st, and developed on Monday. No counts in the gel wash; the protein stain is clear. There is radioactivity in the sample.
Some comment on drying down, retest that, “The gel was too brittle. Next time use glycerol.” But so on, and continuing these tests. 0.04M aluminum [00:15:00] lactate, beautifully defined zone, but badly skewed gel. I must get something, a radiomarker.
More gels being checked in the following pages, alkaline gels, Tris-borate-Versene. Very little migration. [00:16:00] In a TBV buffer, with chloral hydrate. Byron says that pH drops. More radioactive gel being tested. Problems with the gel breaking up on page 51, with a comment that either the gel was overpolymerized, [00:17:00] or should be dried for a shorter time. And tried a vertical gel, “Result indistinguishable, practically from horizontal, I-57, no need to do any more vertical tests. [Try chloroethanol/trichloroethanol?] (inaudible) abandon.” Continuing tests.
[00:18:00] Retest of the alkaline system for the gel, September 5th, but it’s very obviously a poor result. Tritiated isoleucine being checked on September 6th. Labeling and then precipitating with the antibody, anti-beta-2 microglobulin antibody to get out the radioactive beta-2 microglobulin. [00:19:00]Continuing to look at different gels. On Sunday, September 8th, page 69, “Beef heart mitochondria plus NR,” presumably, don’t know what “NR” is. [00:20:00] Multiple bands.
Monday, September 9th, going on with aluminum lactate, and with iodinated insulin chymotrypsin, and DFP chymotrypsin. HeLa I-125 labeling, September 10th, [00:21:00] aim to iodinate reculture, plus anti-beta-2 microglobulin, and recover I-125 in the supernatant. Then, my comments on page 77, my notes on page 77, and presumably Byron’s on page 76. Some little comment on there, that suggests it was Byron, because on the left-hand page, 76, at the bottom, it says, [00:22:00] “The lead shield container was left at Fritz Bach’s lab.” There, that’s where Byron had been unhappy, and had left that lab.
Looking at cells, and [shedding?] of antigen. Get the cells for three hours [00:23:00] under culture. The cells had 31,811 counts of (inaudible) etc., supernatant 171,974, so the cells are shedding label OK. So it looks as if something was getting into the medium, but what it was is not so well-defined here. Set up with an [optical only?] precipitate, and using MDPs, anti-beta-2 microglobulin. And, [00:24:00] testing four counts, and just staining, presumably. Continuing with that total aim. C-14, mix experiment repeated on September 12th, with the comment, however, that the beta-2 [00:25:00] microglobulin does not appear to be labeled.
Antibody precipitation of 125 labeled iodine experiment on page 87. Very slanted gel. Result on Wednesday through Friday. “Exposure shows a trace of beta-2 microglobulin in the supernatant. Gave the solution to Harvey.” That Harvey is Harvey Faber, who was one of the authors of a later paper.
Here is perhaps [00:26:00] the first reference to work with Raju Kucherlapati, because that is how we got together on mapping the chromosome where, on which the gene for beta-2 microglobulin is located. So here, September 16th, Monday, page 91, Raju Kucherlapati describes a QM17 cell line as deployed fibroblast male, with a 16×18 translocation at about the 10th passage, when he sent it to us. Called this latest one, 12QM-17, for the 12th [00:27:00] of September. Should go easily to 20 generations. It can be maintained for at least a month when confluent by feeding twice a week, so this is a cell line from Raju that is making beta-2 microglobulin. That’s interesting how these various friends and collaborators appear at various stages in the notebook and then become lifelong friends, because Raju Kucherlapati is indeed a lifelong friend, as is Byron. [00:28:00] The joy of happy collaborations leading to long-term friendships.
Going back to isoleucine labeling of this new cell line, QM17, essentially confluent, and Byron has some medium labeled, etc., etc. But I — radioactive isoleucine from New England Nuclear. [00:29:00] And, following pages are using it to label the cells.
Wednesday, September 18th, trichloroacetic acid and antibody precipitation of the material from QM-17. And so the gel was run, aluminum lactate chloral hydrate gel, with samples spiked with radioactive iodine, iodinated beta-2 microglobulin, unspiked, and TCA precipitated, and antibody-precipitated [00:30:00] material. But, I was not very happy with the experiment.
The only thing I can see on the film is I-125 spiked marker on the left-hand [00:31:00] side. On the right-hand side, it would appear that the gel has some precipitated, or non-migrating material at the origin. This is #2, described [acid/as the?] film. But anyway, trying to detect the label. A second harvest of isoleucine-labeled QM17, and continued on page 105.
And the next pages, the same material is being considered. With page 111, pretty awful-looking protein-stained gel [00:32:00] with a radioactive autoradiograph showing that there was material at the origin which later then, something which migrated. But, the gel was so awful that it deserves the comment that it got. It was a TBV gel with persulfate, and 0.1% SDS, and poor experiment, a waste of counts. But it was nonetheless autoradiograph, and something could be seen. Whether it was free-labeled or not, there was no comment, just “The gel feels thick, and is glass-hard, and fragmented. Abandon this [00:33:00] type of gel.”
Continuing in the same vein though, riboflavin and TEMED as the different polymerizing agents. Down to aluminum lactate gel, chloral hydrate again on page 125, Thursday, September 26th. A leaky gel tray, etc., etc. Or the gel is not, in fact, too bad. [00:34:00] TBV, SDS again, it didn’t work again.
More gels, page 139, and still having trouble on page 131. “Slots wider than usual due to error in taking off the back glass. Rerun the rest of the sample. Worse still,” is the conclusion from that experiment. The gel looks pretty awful. [00:35:00]
And going on to the experiments, again with the isoleucine label, the only experiment that has worked yet is the I-125 HeLa experiment which had antibody, and on which stripping occurred in two-hour; not established that anti-beta-2 microglobulin, has any effect. Coming to Poulik and Ceppelini, and their method of getting beta-2 microglobulin.
Continuing in the same vein. [00:36:00] First harvest, TCA precipitate, untreated, harvest papain, etc., gels on page 143. So, antibody precipitation of the latest [00:37:00] anti-beta 2M strip. We come to the end of this book. And the results suggests that urea and chloral hydrate are not compatible, and Byron says that chloral hydrate may react with urea.
Gels continue on page 150, on the last page of [00:38:00] this book, [is a pretty heavy?] label of Byron’s Book b, page 24, HeLa membranes 550,000 counts, of the gel, and the autoradiograph is very heavy at that point. However, there are also two samples, one with I-145 control, I-125 beta-2M-spiked medium, where there’s a clear band, and then, that’s the sample being 41,000 [00:39:00] count of control, new I-125 beta-2M carrier, so the beta-2M can be seen in these gels, but there is no good evidence that we’ve been successful in labeling it yet. And that is the end of Book i.