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??)