Hi all,
Currently working on in-gel digestion for peptide identification, other than using ziptip or centrifugal unit to desalt sample, I heard using TCEP also works well to desalt.
I wish to understand more about it, anyone can share their knowledge?
Highly appreciated
My experiences in thiol-reducing agents suggest that 2-Mercaptoethanol/ME is the best (please see file; Thiol-type BIN). Further, in order to protect the Cysteine residue, the pyridylethylation (PE-derivatiztion) seems to be best and stable (please see file; PTH Roma RP-HPLC).
I am now considering that too strong is not so good, as famous Chinese Philosopher Confucius (China; BC 6 C: 孔子) has already declared previously, is surely the truth (please see file; Feed by Measure). Similar idea has been also said by Aristotle (Greece; BC 4 C) and Gotama Siddhattha (India; BC 6 C).
By the way, the ancient Chinese-Emperor of Shi Huangdi (Qin dynasty; BC 3 C), who believes the “Taoism” and finds “the elixir of immortality by mercury”, may have possessed a good intuition.
Further, active center of biotinidase shows strong Species differences; i.e., Porcine kidney biotinidase is thiol-type (as indicated by famous German Biochemist Dr. Joachim Knappe), but Bacterial enzyme is not (indicated by both Dr. J. Knappe and Dr. H.G. Khorana). We also has reported that Rat kidney biotinidase is thiol-type (please see file; J Chrom B Rat BIN LIP Km).
Human serum biotinidase has been firstly shown to be the glycoprotein enzyme conaitining N-Acetyl-Neuraminic acid/NANA by Dr. Krishnamurti Dakshinamurti (Winnipeg, Canada), but he has denied the contribution of glycochain of biotinidase on enzyme kinetics. This may be due to utilization of non-quantitative direct-utilizatin of photometer in his biochemical studies. Thus, enzyme assay should be performed by reliable and sensitive HPLC-photometric enzyme assay method (please see file; BIN Detr BPAB). The effect of glycochains of liver biotinidase has been shown by using reliable HPLC-photometric enzyme assay method (please see file; Anticancer Res). The effect of glycochains of human serum biotinidase has also been reported by using reliable HPLC-photometric enzyme assay method (please see files; Wide range of Biotin and JMBT Alopecia).
Hi,
TCEP is Tris(2-carboxyethyl) phosphine, a reducing agent used to reduce cysteine-cysteine disulfide bonds in proteins. TCEP usually does not need to be removed by desalting prior to further thiol (-SH) reactions with Iodoacetyl reagents.
For protein identification by peptide mass fingerprinting I use DTT and IAA, respectively for reduction and alkylation of cysteines, as described by
Article A simple and efficient approach to improve protein identific...
Hi Tang Zhi Zhuang,
Ziptip used to desalt less than 100 ug proteins. If you have more than 100 ug proteins (starting amount), you should use Sep-Pak C18 cartridge (Waters, Milford, MA) column for desalting. it is very convenient and easy to handle. You can find the protocol here- https://www.ccmp.ox.ac.uk/_asset/file/sep-pak-c18.pdf
Hope this works.
Hi Tang Zhi Zhuang
In our lab, we generally prefer to desalt using Empore C18 material with Solvent as [0.1% Formic acid] and elution by Solvent B [40% Acetonitrile (ACN), 0.1% Formic acid]. We also use different concentration of TFA as Solvent A with elution using Solvent B [40% Acetonitrile (ACN), liquid ammonia].
Varsha
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