slides. Staining of section with 0.1% toluidine blue in 1% borax, pH 11 was done for general structure observations. To determine the location of starch deposits in cotyledons of ungerminated and germinated PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645759 seeds, semithin sections of seeds were stained with I2-KI solution. Slides were observed under light microscope. Immunohistochemistry Slices of cotyledon tissue were fixed in 3% glutaraldehyde in 0.2 M phosphate buffer, dehydrated and embedded in paraffin wax. For light and fluorescence microscopy, sections were prepared using Leica RM2245 semi-motorized rotary microtome. Approximately 6 mm thick sections were cut and collected on polylysine coated slides. Prior to staining sections were deparaffinised using xylene and rehydrated using 100%, 90%, 70%, 50% and 30% alcohol, respectively in sequential order, for 5 min each. Sections were pre-incubated in 5% BSA in PBS at pH 7.4 for 30 min, then incubated for 4 h in primary antibody diluted in PBS containing 1% BSA and 0.32% Tween 20 at 1:500 dilution. Slides were rinsed four times for 15 min with PBS-Tween, then incubated for 2 h, in the dark with secondary antibody -FITC conjugate) diluted 1:300 in PBS, 1% BSA, 0.32% Tween 20. Slides were then rinsed 4 times with PBSTween for 15 min each. Longitudinal sections were analysed Results Purification Crude extract was prepared from ungerminated seeds, as described under experimental section. The fractionation of proteins by acetone followed by anion-exchange chromatography using DEAE-cellulose and glycogen precipitation, as outlined in table 1, resulted in purification of Fenugreek b-amylase 210 fold with a yield of 21%. Homogeneity of the sample obtained after glycogen precipitation was checked on SDS-PAGE, which revealed a single band after CBB staining. Mr of enzyme 
was determined to be 58 kD, when compared to MedChemExpress Neuromedin N standard molecular weight markers. Activity staining showed development of clear band over a dark background b-Amylase from Starchless Seeds of T. graecum formed due to complex of starch and I2-KI solution, corresponding protein band on Native-PAGE is shown in Fig. 1 B. The purified protein gives a single peak from Sephacryl S-200 gel filtration column and the molecular mass of native Fenugreek bamylase determined from the mean of two experiments with it was 56 kD, which is in accordance with the mass obtained on SDSPAGE, hence suggesting monomeric nature of enzyme. pI of Fenugreek b-amylase was determined to be 5.2 from IEF. The peaks obtained after MALDI-TOF analysis and the peptide mass fingerprints were used for database searching using MASCOT. 7 b-Amylase from Starchless Seeds of T. graecum The most significant match was found with b-amylase of Medicago sativa with a score of 82 and Evalue of 0.0015. Characterization of Fenugreek b-amylase Enzyme hydrolyzed starch at highest rate followed by amylopectin, amylose and glycogen. Pullulan was not hydrolyzed indicating that enzyme failed to cleave a-1,6 bond. Also, the inability to give colour with assay performed using starch azure as substrate suggested that it was endoamylase. Fenugreek b-amylase was not able to degrade native starch. There was no loss in enzyme activity after dialysis against buffer containing 5 mM EDTA for overnight, which indicates that Fenugreek b-amylase is not a metallo-enzyme. The characteristic property of b-amylase to release exclusively maltose as end product of starch hydrolysis was identified by paper chromatography. Periodic acid-Schiff’s reagent me