Ideas
1- Dansylation of small protein as TCA carrier
2- Effect of salts on TCA ppt, better stain for Sarkosyl pellets (Sudan?)(GOOD)
3- Dissolve TCA pellets with reduced amounts of Tris
4- Secretome - pellet Chlorella and suspend in small volume (GOOD)
5- Glycosidase activity in secretome? (NAG yes)
6- Silver stain gels of soluble and WAL fractions
7- Extended proteinase K treatment (high T, SDS)
9- Does lysin produce CHO fragments which inhibit?
10- Add volatile base to THF to neutralize TCA (TEA problematic)
11- Purify WAL lysin w/o reducing agent (GOOD)
12- Ammonium sulfate fractionation of WAL lysin (IN 30-65% FRACTION)
13- Can ammonium sulfate extract cellulase, etc. from virions? (EXTRACTS LYSIN)
14- Do detergents inhibit glycosidase activities?
15- Can WAL lysin digest walls C169 (NO), PBi(YES)?
16- Can ether extract 4MU from 4MU-phosphate, 4MU acetate?
18- Acid sensitivity of lysin (RESISTANT!)
19- Radial diffusion assays of soluble lysin preps
20- Purify lysin from fuzzy plaque variants
21- Fluorescent reducing sugar assay
22- Does Y-Per have lysin activity? (NO!)
23- Develop small protein carrier
24- Run lysin through a second column
25- Run lysin over cation exchanger (STICKS STRONGLY!)
26- Do zwitterions affect lysin activity?
27- Mass spec on gel fractions
28- Gel filtration of lysin?
29- Salt series for extraction lysin, RT, 37C, + DTE
30- Can acids extract lysin? (YES!)
31- A215L activity? Longer incubations? (YES!)
32- Calcafluor to monitor cells
33- Effect of antibodies on lysin
34- Optimize A215L assay (time, temp, salt)
34- Add other cloned genes to A215L
35- Sonication - series of sonication times (SONICATION UNNECESSARY)
35- Sonication of cloned A215L with and w/o added salt (SAME)
36- Sonication to remove tail
37- Virion sonication - effect of pH (SALT ALONE WORKS WELL)
38- Shorter sonication times incubate at higher T
39- Incubate aliquots of lysin extraction at higher temps
40- Wash lysin from virions with high salt (virion disruption?)(1M LiCl WORKS WELL)
41- Clean up alginate lyase with ammonium sulfate (ALSO PPTS JUNK)
42- Protein gel of salt solubles from virion
44- Antibody to A140/145R (PAPK)
45- Radial diffusion with alginic acid
46- Degrade virus with EDTA, DTE to get lysin (INEFFECTIVE, DTT INHIBITS)
47- LiCl treatment at lower pHs
48- Alginate lyase activity on SAG cells (ACTIVITY SPECIFIC TO PBi)
49- Alginate lyase activity at higher pH (BETTER AT 9 THAN AT 8)
50- Artificial lysin, variety of cells, conditions (NONE AS GOOD AS ALGINATE LYASE)
51- Screen complete cell set with artificial lysins (MINOR ACTIVITIES)
52- Does lysozyme inhibit lysin?
53- Are there autolysins?
54- Effect of Ca, EDTA, Temp on alginate lyase (EDTA SENSITIVE)
55- Assay lysin by nucleic acid release (SYBR GOLD LOOKS GOOD)
56- Optimize Sybr Gold assay, 600nm filter, lower dye, cleaner samples (DARK READER ORANGE WORKS WELL)
57- Sybr Gold with E. coli, lysozyme
58- Bacterial colonies that use Chlorella as carbon source.
59- Photogenic lysin, lyase assays (OK)
60- Purify lysin with pelleting of virus, acid extraction
62- Does lysin assay work with lower cell concentrations?(YES)
63- Cationic dye to monitor cell levels?
64- Extract lysin from purified ATCV with LiCl(SPECIFICITY AS PBCV)
65- Sybr Gold assay of alginate lyase at higher enzyme levels (WORKS WELL)
66- DEPC concentration series (inhibitor)
67- DTT concentration series (inhibitor)
68- Extract A215L with LiCl, acid (pH 5 W/LiCl WORKS BEST)
69- Do peroxide, dihydroxyethyldisulfide restore DTE inhibited enzyme?
70- Proteinase K on Chlorella panel (EDTA, sarkosyl, SDS), check with Irina
71- Effect of Calcium ion on lysin with Chlorella panel
72- Sybr Gold assay for PBCV stability
73- Sybr Gold for clearing time
74- Cellulase, etc at higher T, Sybr Gold assay
75- Sybr Gold through orange filter (AOK)
76- Lysin activity with Chlamy
77- Extract lysin at pH 4.5 (AOK!)
78- NC64a, SAG assay blanks with alginate lyase (LYASE ACTIVE)
79- Proteinase K digestions (10mg/ml x 4hrs) (NOT ABOVE BKG)
80- Ammonium sulfate extraction, purification of PBCV1 lysin
81- Pellet LiCl treated virus, look at supernatant proteins
82- LiCl concentration series on standard lysin assay (0.2m INHIBITS)
83- Follow lysin digestion by microscopy
84- Two phase lysin assay? Chlorophyll in ether, chloroform, butanol?
85- Column chromatography of LiCl extract - lysin assays
86- DNase treatment of LiCl extracts (DNase DOESN'T LIKE HIGH SALT)
87- Calcafluor to follow lysin activity
88- PBCV dissociation in the presence of IAm
89- Sucrose gradient on salt dissociated virus (need glutaraldehyde??)
90-