Friday, February 28, 2020
Protein Misfolding Amyloid Lab Report Example | Topics and Well Written Essays - 3000 words
Protein Misfolding Amyloid - Lab Report Example Fig 3. Fluorescence intensities of a) Tyrosine b) Tryptophan and c) Thioflavin T in Con A , at 40 0C a) b) c) Fig 3 b also shows considerable Trp fluorescence at pH 5.0 and 7.0 while at pH 9.0, intensity remained insignificant again showing buried/ quenched Trp residues. After a lag of 40 h the ThT intensity increased sharply to high level at pH 5.0. At pH 7.0, the plateau was attained after slight increase up to 20 h. At pH 9.0 the intensity increased at 70 h. Amyloid A40 Assay: Fig 4. Fluorescence of a) Tyrosine and b) Thioflavin T in A40 at 250 C Fig 4 a) The ThioflavinT bound to A increases at 40 h decreases thereafter and increases indicating conformational changes. Fig 4 b) Fig 4. shows Tyr intensity decreasing after 60h and so is the Th T intensity however the latter increases thereafter showing increased amyloidal fibril formation but at this time the Tyr seems to be quenched somewhat. Fig 5. TE Micrographs of Con A in pH 5.0 at 0 h Fig 5 a) X10K some amorphous aggregates are already present at this pH Fig 6. TEM showing Con A in pH 5.0 at a) 4 (X40K) and, b) 48h (X40K) c) 48 h at pH 7.0 (X15K and d) 48h at pH 9.0 at 25 0C Fig 6 a) only amorphous aggregates are present Fig 6 b) Fibril formation is clearly visible. Fig 6 c) 48h at pH 7.0 (X15K) Fig 6 d) 48 h at pH 9.0 (X10K) as expected the long amyloid fibrils are in state of formation Fig 7. Con A at 37.2 0C and pH 5.0 after 24h The physiological temperature and low pH shows abundant short rods Fig 8. A40 at 25 0 C a) 0, and b) 3h. (X20K) neither shows fibrils despite positive ThT fluorescence. Fig 7 shows AB40 as small...But at pH 9.0 the intensity decreased sharply from beginning up to 70h and then attained a plateau. The decrease of 400u indicated huge conformational change leading to buried and /or quenched Trp residues. Th T fluorescence decreased slightly up to 50 hrs and increased sharply thereafter reaching at peak at 80 hrs and then decreasing considerably. At pH 7 the intensity increased from beginning, reaching a max at 50h and decreasing sharply, thereafter. The Th T fluorescence shows reversible trend in these experiments and conformational changes are occurring fast. At pH 9.0, there was considerable increase in fluorescence intensity after 75h showing fibrillation (fig 1 c). Highest intensity was observed at pH 5.0 while considerably high Intensity at pH 7.0. However at pH 9.0 there was negligible intensity for tyrosine. The latter temperature and pH combination either create conditions for Tyrosine quenching or the aggregates deeply bury this amino acid as a result of conformational changes (Fig 3a). Fig 4. shows Tyr intensity decreasing after 60h and so is the Th T intensity however the latter increases thereafter showing increased amyloidal fibril formation but at this time the Tyr seems to be quenched somewhat. Only large amorphous structures seen after 72 h while a solitary long fibril is seen in the upper region after 96h (Fig 8 b).