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Why is it necessary to cleave proteins with at least two
different reagents during sequencing?
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Answer / quido
generally the protein or polypeptide to be sequenced needs
to be cleaved into segments of size suitable or amenable to
the sequencing technique.
2 different reactions with two different cleaving agents is
used in order to allign the segments correctly once
sequencing is completed, with the help of overlapping
sequences, from the two digests.
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I have given the protocol for the cyclodextrin glygosyl transferase assay: One ml of appropriately diluted enzyme sample was incubated at 60 °C for 15 min with 5 ml of 1% (w/v) gelatinized soluble starch in 50 mM, 7.0-pH Tris–HCl buffer. Reaction was terminated by boiling the reaction mixture for 3 min and reaction volume was made to 10 ml with distilled water. Two ml of above reaction mixture was withdrawn and mixed with 3 ml of Tris–HCl buffer, 5 ml of 125 mM Na2CO3, and 0.5 ml of phenolphthalein (25 mg phenolphthalein/100 ml absolute alcohol). Absorbance was measured at 550 nm. The percent decrease of sample was calculated with respect to control containing 5 ml of buffer, 5 ml of sodium carbonate and 0.5 ml of phenolphthalein. where Acontrol = absorbance of control and Atest = absorbance of sample. The amount of β-cyclodextrin (β-CD) produced was estimated from the standard graph of 0–500 μg/ml β-CD concentration against % decrease in absorbance. One unit of CGTase was defined as the amount of enzyme required to produce 1 μm of β-CD/min. Please can you suggest me the formula for the defination given in the last line
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