I want to check the serum and protease stabilty of a cationic peptide can I go with native gel electrophoresis other than HPLC. Just to see fragmentation if it's not stable. If someone have done Native gel electrophoresis to check stabilty of any protein in serum.
I think it would be very difficult to monitor stability of a peptide in serum using gel electrophoresis, either native or denaturing, because of the enormous quantity of protein in serum. You could monitor the protease stability more easily this way as long as autoproteolytic products of the protease don't obscure the peptide. If the peptide has a fluorescent dye attached, the experiment might be easier because you would not have so much background from the other proteins in serum or the autoproteolytic products or the protease.
Thanks for ur valuable suggestions. So may I go with HPLC??? As I had seen such research papers checking the stabilty by HPLC
http://www.ncbi.nlm.nih.gov/pubmed/18726574
These guys had checked serum and pretease stabilty of peptide by HPLC
Actually I had seen this paper which made me to think like that....
Stability of amyloid-β peptides in plasma and serum.
Bibl M1, Welge V, Esselmann H, Wiltfang J.
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Abstract
Plasma amyloid-β peptide (Aβ) levels have been suggested as a biomarker candidate for detecting incipient AD. Aβ peptides are known to be sensitive to distinct preanalytical sample handling, which calls for standardised preanalytical procedures. We investigated serum and plasma samples of 19 patients with no clinical signs of dementia for different preanalytical sample handlings. Both serum and plasma were analysed by the one-dimensional Aβ-SDS-PAGE/immunoblot, either immediately or after storage at room temperature for 24 and 48 h, respectively. The panel of Aβ1-37/38/39/40/42 and Aβ2-40 was evaluated. In both analytical matrices, sample storage led to a significant loss of measurable peptide levels. This effect was most pronounced during the first 24 h of storage and stronger in serum than in plasma. There were no significant differences between the distinct analysed Aβ peptide species regarding these results. The ratios of peptides (e.g. Aβ1-42/Aβ1-40 and Aβ1-42/Aβ1-38) displayed a higher stability under the influence of storage than each single peptide. In conclusion, plasma may be more appropriate than serum for analysing Aβ peptides for routine application. At least, the analysis should be done within 24 h and peptide ratios should be created to minimise artificial results.
The abstract says that they used SDS-PAGE/immuoblot, in other words a Western blot. For this you require an antibody that recognizes the peptide. You also have to be able to resolve the peptide on the SDS-PAGE gel, which might be challenging if the peptide is very small, and find suitable conditions for transfer to the membrane.
Yes So that will very tedious work....So It seems HPLC or Mass spec is the only option left....As these guys Hansen et al suggests. Thanks Sir for ur kind response.
http://www.ncbi.nlm.nih.gov/pubmed/18726574
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