I'm having difficulty standardizing a method for extraction and isolation of peptides from plant seed and fruit pods.
Dear Sir. Regarding your issue about the method for extraction and isolation of peptides from plant seed and fruit pods. For extraction of proteins (peptide) from plant materials. The seeds were dried to reduce 10% moisture at 600C. These samples were kept at 40C in a tightly closed polyethylene bags before use. Dried seeds were ground with liquid nitrogen using pre chilled motor and pestle and Phosphate buffer pH 7.5 .The supernatant was further salt precipitated, cutoff separated and used for the estimation of total protein. The sample was centrifuged at 10000 rpm for 15 min and stored at -20 in a freezer. Partial purification of small peptides is carried by Ammonium sulfate precipitation phosphate buffer and liquid nitrogen processed samples containing soluble portions were treated with 75% ammonium sulfate maintained at 40C for 20 h and centrifuged at 10K rpm for 20 minutes. Precipitated proteins were further purified by buffer exchanged with 0.1M phosphate buffer by stirrer. Protein concentration was estimated after ammonium salt precipitation. Cut off separation The dialyzed protein samples were passed through 5 kDa cutoff spin column. The flow through obtained after centrifugation at 5000 rpm for 10 min was the protein. Estimation of Total Proteins by Lowry’s method seeds extracted mixture, ammonium sulfate precipitated samples and 5Kd cutoff flow through were used for estimation of Protein content by Lowry method using bovine serum albumin (BSA) as standard. The experiments were repeated in triplicates. For more details, the following below attached file and links may help you in your analysis:
http://www.pakbs.org/pjbot/PDFs/39(1)/PJB39(1)211.pdf
http://www.pakbs.org/pjbot/PDFs/43(6)/54.pdf
Thanks
Dear Amanda, for extraction and isolation of peptides from plant seed, you can go through the following link. it may help you for low molecular weight peptides isolation.
http://innovareacademics.in/journals/index.php/ijpps/article/viewFile/9691/3540
can any body help me to tell what is the best method of low molecular weight peptides isolation from milk
will this same suggestion will be suited for plant leaves also
And i have a doubt
what is the difference between the use of fresh leaves and the use of liquid nitrogen frozen leaves
Dear
Based on my experiences, ultrafiltration using membrane 10 kDa for size limitation and 1 kDa for condensation will help you for isolation of small peptides. However, further purification with HPLC will also be necessary.
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