Ideas 5
Ideas 5

1- Attachment assays with microsocope

2- Factors affecting virion fluorescence

3- Extract lysin from virion with acid, gel filtration

4- LiCl proteins, SDS proteins, total proteins from NC64a

5- Virus prep at lower pH - plenty stable

6- Effect of protamine sulfate on virus purification - AOK

7- Effects of EGTA, Ca on virus purification

8- Purify with CPC (OK but "lingering ppt")

9- Heat treatment during purification (withstands 50C at pH 5, 4.5)

10- n-butanol, extract with iso-butanol or octanol

11- Incorporate trypsin, chymotrypsin into purification? - (pepsin works)

12- Quantitative lysin assays with Janet's strains

13- Isozyme gels

14- Binding assay - wash cells

15- Protein gels on virus preps (all look the same)

16- Sucrose gradients of virus preps

17- Repeat virion salt dissociation, assay on PBi (virus binds to PBi)

18- Nile red staining of virions

19- Ratio of Sybr Gold to virus

20- Kinetics of Sybr Gold binding to PBCV

21- Effect of SDS on Sybr Gold binding to PBCV (suppresses)

22- Effect of salt conc. EGTA, Ca++ on Sybr Gold binding to PBCV

23- Effects of DNAse, chymotrypsin on Sybr Gold binding to PBCV

24- Cell counts and PBCV quantitation

25- Effect of salt on lysin activity and virion fluorescence (lysin works, 1ul of 10x dil 2M LiCl)

26- 37C, 42C effects on salt dissociation

27- Ammonium sulfate ppt of virion

28- Protease treatment of NC64A cells

29- Glycanase treatment of virion

30- What's required to extract chlorophyll from ATCV?, Triton, sarkosyl, pepsin, DTT?

31- pH stability studies - pH shift to see fluorescence of acidified virus (stable to pH 4.5)

32- Activity of ATCV lysin - comparison w/protein gels

33- Amylase for chlorovirus purification

34- Effect of higher pH, Triton on lysin extraction from virions

35- Virion proteins after salt extraction

36- Protease sensitivity of virion in the presence of salt - sybr gold assay

37- Effect of chymotrypsin on salt treated PBCV - 37C v 42C, EGTA v Ca