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