Medical Research Council notes, 1971-1972
Medical Research Council notes, labelled 'Book 4/P3' - P3 refers to Potter tumour 3, Jun 1971-1972:
- Experiments optimizing conditions for an antibody assay for P3 IgG using model IgG kappa chain and antibodies from Cesar Milstein and his student Svasti (15/6).
- "A" refers to an initial zonal RNA separation. "A, re-run" is a re-run of sucrose gradient fractions of RNA ("A") isolated from P3 solid tumours (the neater writing is that of research assistant, Elma Cartwright), 17 Jun 1971.
- Appears to be the first experiment with rabbit reticulocyte (retic.) lysates (from Tony Hunter, Biochemistry Cambridge) assaying for 35S methionine incorporation into TCA precipitable counts. Tube 12 gave increased cpm incorporated in response to added mRNA, over the controls (tubes 3, 4), 18 Jun .
- [The experiment reported as Fig 1 in Milstein et al (1972) is apparently not in this notebook - it may have been in Elma Cartwright's notes since this experiment was first done by her although Brownlee remembers marking the radioautograph with the position of the unlabelled light chain added as a marker. This was also the first time the team ran the Laemmli modification of the Weber and Osborn acrylamide gels, which gave improved resolution. No one had done this before for retic lysates believing (incorrectly) that the large excess of haemoglobin would overload the gels.]
- Another mRNA preparation “B” from Potter P3 tumours followed by 3% polyacrylamide gels (? stained by acridine orange). TH is Tim Harrison, Brownlee's first PhD student, 21 Jun 1971.
- "D" is another zonal mRNA preparation ("C" was lost on the zonal centrifugation), 27 Jun 1971.
- In both Brownlee's writing and (neater) that of Elma Cartwright Gurdon (John Gurdon) is mentioned. Brownlee thinks the idea was to give him mRNA to translate in frog oocytes, 30 Jun 1971, 1 Jul 1971.
- Reference to Stavnezer and Huang’s (1971) paper, 14 Jul 1971.
- Making up -cysteine, - methionine ad – isoleucine, amino acid mixtures (in Elma Cartwright's handwriting).
- Test of new retic. lysate batch. Results not shown, 16 Jul 1971.
- Test of 14C amino acid mixture and retic. lysate titrating haemin and time course. Also test of 14C Ileu & 35S met versus concentration of added label and haemin concentration, 19 Jul 1971.
- Myeloma mRNA with 14C Ileu with various A and D mRNA preparation and retic lysate (19-3). After incubation, assay by a direct precipitin reaction. Not conclusive, 20 Jul 1971.
- (With Mike Mathews) using samples 2 & 3 of prep D. Also give to TH (Tim Harrison) to run SDS gels, 23-24 Jul 1971.
- Prep "E", fractions 1-4 as usual and RNA gel analysis comparing preps A, B, D & E, 26 Jul 1971.
- 30 Jul 1971: Test of Prep D using Mike Matthew ascites cell extracts and 14C amino acid mixture. Large scale experiment. 6 Aug 1971 conclude from TCA counts that fractions A & D were positive (both Brownlee's writing and Elma Cartwright's). Ran SDS gel 11 Aug 1971 (unknown handwriting).
- Another large scale synthesis of 35S labelled translation products of mRNA fractions (preparation D) using the Mike Matthews S30 extracts. Elution of protein from SDS gels (with PMSF & carrier protein). Give to CM (Cesar Milstein) for peptide analysis, 12 Aug 1971.
- Another large scale synthesis (1 ml) of 35S labelled translation products of mRNA fractions (preparation D) using the Mike Matthews S30 extracts. Run SDS gels with IgG marker (from Tim). Unsuccessful. 20 Aug 1971.
- Prep E test in mouse ascites extract (MM - S30) with 14C amino acid mixture. Positive, 18 Aug 1971.
- Different RNase inhibitor concentrations, 1 Sep 1971 and 7 Sep 1971.
- Another large scale translation of 35S labelled kappa chain for peptide analysis by Cesar M. Positive, in giving bands on pH 3.5 electrophoresis, 6 Sep, 23 Sep 1971.
- Prep F, 4 Oct 1971.