Lipotso Tse Botsoang Khafetsa

Ho ea ho Complex ho tloha ho limiligrama ho isa ho lik'hilograma


Kopo ea Quote e Tloaelehileng


How to store peptides?


Li-peptide tsa Lyophilized

Bakeng sa polokelo ea nako e telele ea li-peptide, re khothaletsa ho li boloka e le phofo e tiileng. Li-peptide tsa Lyophilized li ka bolokoa ho -20 ° C kapa ka tlase ka ho senyeha ho fokolang. Bareki ba eletsoa ho hlahloba hape li-peptide tse ikhethileng nako le nako 2 lilemo ho tloha ka letsatsi la tokollo ea taolo ea boleng.

Li-peptides ka tharollo

Li-peptide ka tharollo ha lia tsitsa ebile li na le tšekamelo ea ho senyeha. Litharollo tse nang le metsi tse sebelisoang ho qhala li-peptide li lokela ho ba tse sa hloekang le tse hloekileng.

Ka tharollo, tekanyo e itseng ea peptide e ka senyeha, ho itšetlehile ka li-amino acid tse teng ka tatellano. Mehlala e meng e thathamisitsoe ka tlaase mona:
– Li-peptide tse nang le methionine, masala a cysteine ​​kapa tryptophan a lokela ho ba le nako e lekanyelitsoeng ea ho boloka tharollo ka lebaka la oxidation. Li-peptide tsena li lokela ho qhibiliha ka har'a motsoako o se nang oksijene. –
Glutamine le asparagine li ka felloa ke matla ho glutamic acid le aspartic acid, ka ho latellana. –
Cysteine ​​​​e ka kena ho oxidative cyclization ho theha bond ea cysteine-cysteine ​​​​disulfide (e ka bopjoang ka hare kapa pakeng tsa ditlamo tsa disulfide). –
Masala a lefisitsoeng (asiti ea aspartic, asiti ea glutamic, lysine, arginine, histidine) li na le hygroscopic (ho monya metsi moyeng) le tšekamelo ea ho bopa viscous, oli e hlakileng. Phetoho ena ea 'mele e kanna ea se ame sebopeho sa peptide. Ho khothalletsoa hore li-peptide tse joalo li be lyophilized ho tlosa palo ea metsi, ebe e tšeloa ka naetrojene ho netefatsa hore ha ho na mouoane oa metsi ka har'a vial, ebe o koaheloa ka potlako.

Ho thibela tshenyo hore e se be le serame kgafetsa le ho qhibidiha, re khothaletsa basebelisi ho qhala feela palo ea peptide e hlokehang bakeng sa teko ea hajoale. Tharollo e feteletseng e lokela ho bolokoa ho ≥ -20 o C ho fihlela ho hlokahala.

Qoba mongobo.
Kaha mongobo o fokotsa haholo botsitso ba nako e telele ba li-peptide, re khothaletsa ho leka-lekanya li-peptide ka har'a desiccator ho mocheso oa kamore pele u bula linkho. Kamora ho fana ka peptide, li-peptide tse setseng ka har'a tube li lokela ho hloekisoa ka bonolo ka nitrogen ea anhydrous, joale setshelo se lokela ho koaheloa, e tiisitsoeng ka filimi e tiisang, 'me e bolokoe ho -20°C.


Litšobotsi tsa ho qhibiliha ha li-peptide tse fapaneng li fapana haholo. Mesaletsa e kang alanine (Ala), cysteine (Cys), isoleucine (Ka), leucine (Leu), methionine (Kopana), phenylalanine (Phe), le valine (Val) eketsa ho hydrophobicity ea peptide le ho qhibiliha ka har'a litharollo tse nang le metsi, 'me ho khothaletsoa hore moreki a latele litataiso tsena ha a etsa li-peptide.

Thepa ea Solubility

Ho qhibiliha ha li-peptide ho itšetlehile haholo ka tatellano ea amino acid. Ka tlhaho li-peptide tsa hydrophobic (A, F, G, V, L, I, M, W, P e nang le tšekamelo e phahameng) hloka lihlapolli tsa manyolo bakeng sa solubilization. Li-peptide tse nang le asiti (tšekamelo e phahameng bakeng sa D, E ka tatellano ea peptide) hloka li-buffer tsa motheo tse nang le metsi bakeng sa solubilization, ha li-peptide tsa motheo (tšekamelo e phahameng bakeng sa K, H, le R) hloka acidic aqueous buffers bakeng sa solubilization.

Khetho ea Solvent

Ho latela mefokolo ea tlhahlobo ea hau, re khothaletsa ho sebelisa litataiso tse latelang ho fumana hore na solvent e ntle ka ho fetisisa ea solubilizing peptides

Li-Peptides tsa Hydrophobic

Ho kopanya hape li-peptide tsa hydrophobic, eketsa 100 µL ea DMSO le sonicate ho fihlela ho etsoa tharollo ea homogeneous. E 'ngoe, eketsa buffer ea khetho ea hau ho etsa a 1 tharollo ea mg/mL (e phahameng ea khatello ea peptide, boholo ba palo e hlokahalang ea DMSO).

Hydrophilic (E nang le asiti) Li-peptides

Ho tsosolosa li-peptide tse nang le asiti, eketsa 100 µL ea 1% NH 4 Oho ho 1 mg ea peptide le vortex. Hang ha tharollo e hlakile, eketsa buffer ea khetho ea hau ho etsa a 1 tharollo ea mg/mL.

Hydrophilic (Motheo) Li-peptides

Ho theha bocha peptide ea mantlha, eketsa metsi a distilled ho 1 mg ea peptide le vortex.


Tekanyetso ea indasteri ke ho fana ka li-peptide ka sebopeho sa lyophilized joalo ka Boima ba Gross. Boima bo tebileng ba peptide ke boima ba kakaretso ea likarolo tsohle tse teng ka phofo ea lyophilized. Sena se kenyelletsa peptide ea thahasello, litšila life kapa life tsa peptide, metsi, metsoako e setseng, le counterion.

Ho fapana le moo Nete boima ba peptide, e baloang ho tloha ho Likahare tsa peptide tsa Net, ke boima ba ke peptide feela karolo e teng ka phofo ea lyophilized. Sena se fana ka lintlha tse nepahetseng haholoanyane tsa tharollo ea hau ea li-peptide tse latelang. Kahoo, re khothalelitse boima ba 'mele ba peptide bakeng sa Peptide e tloaelehileng ditaelo.

Net Peptide Content Papiso

Net Peptide Content tekanyo e etsoa ka tlhahlobo ea amino acid (AAA; ho nepahala ho fokolang empa ho hloka thepa e fokolang) kapa tlhahlobo ea mantlha (CHN; e hloka limiligrama tsa peptide empa e nepahetse haholoanyane). Mekhoa ena ka bobeli e fana ka boleng ba peresente (ke hore., 75% litaba tsa peptide).

Net Peptide Content e ipapisitse le palo ea likhakanyo tse teng *, e itshetlehileng ka tatellano ya peptide. Ha e le hantle, tefiso ea peptide e phahame, e fokotsa maemo a peptide

*Hlokomela: Ntho e sebelisoang ho bala net peptide content e ipapisitse le peptide counter-ion. Sena se etsoa ho netefatsa hore counter-ion ha e kenye letsoho ho palo ea peptide. Ka mohlala, ha TFA kapa acetate e le counter-ion, naetrojene e sebelisoa hobane e fumaneha ho peptide empa eseng ho TFA kapa acetate. Carbon kapa Nitrogen e ka sebelisoa ha chloride e le counter-ion ka lebaka le tšoanang.

Lipalo tsa Net Peptide Weight:

Net Peptide Palo (peptide e felletseng) - mokhoa oo re o sebelisang bakeng sa liodara tsa peptide tse tloaelehileng.

Eketsa Peresente ea Net Peptide Content (foromo ea decimal) ka Peptide Gross Weight, ho fumana palo ea Net Peptide Quantity (peptide e felletseng).

Ka mohlala:
Bakeng sa 5 mg gross peptide/vial:
Likahare tsa marang-rang tsa Peptide ke 84%.
Palo e nepahetseng ea kakaretso ea peptide = 0.84*5 mg=4.2 mg kakaretso ya peptide

Net Peptide of Interest Quantity-e nepahetse ka ho fetisisa - moreki a ka sebelisa mokhoa ona ka bobona ho bala peptide ea thahasello.

Atisa Peresente ea Net Peptide Content ka Peptide Gross Weight, le Peptide Purity (ka HPLC), ho bala palo e nepahetseng ea peptide ea thahasello eo u nang le eona. Liphesente tse ngata tsa peptide (foromo ea decimal) e atisoa ke bohloeki ba peptide (foromo ea decimal).

Ka mohlala:
Bakeng sa 5 mg gross peptide/vial with:
Likahare tsa marang-rang tsa Peptide ke 84%.
Palo e nepahetseng ea peptide ea thahasello ke = 0.84*0.96*5 mg=4.03 mg


Liphetoho tsa MOL li sebelisa k'hemistri ea Fmoc e tiileng ho fapana le k'hemistri ea BOC. Fmoc chemistry e lumella ho tlosoa ha lihlopha tse sireletsang ho sebelisa maemo a bonolo nakong ea ho lelefatsa peptide, ha k'hemistri ea BOC e hloka ho sebelisoa ha acid e matla ho tlosa lihlopha tse sireletsang nakong ea ho lelefatsa peptide. Ho phaella moo, Fmoc chemistry e sebelisa trifluoroacetic acid (TFA) ho tlosa peptide ho resin, ha k'hemistri ea BOC e sebelisa hydrofluoric acid (HF). HF ha e na 'mala, e senyang haholo, le chefo ea ho kopana.

Ka hona, Liphetoho tsa MOL li sebelisa k'hemistri ea Fmoc kaha e bonolo haholoanyane, tenyetsehang, 'me e sebetsa ka mefuta-futa ha e bapisoa le k'hemistri ea BOC.


Molarity e bolela mokoloko oa molar oa tharollo, e leng palo ea li-moles tsa solute tse qhibilihisitsoeng ka har'a 1 litha e le nngwe ea tharollo, e hlahisoa joalo ka mol/L, kapa M.

Molarity [M] = 'Misa / (Molumo oa x Molar Mass); Mole = Ho tsepamisa maikutlo (g/L) x bophahamo ba modumo (L)/MW (g/mol)

Mohlala:
Fuoa: 1mg ea phofo ea peptide e omileng e nang le MW: 20KDa (Molar boima ba peptide ke 20 g/mol)
Ho fumana molarity ka boima bo tsebahalang le bophahamo ba modumo
Bakeng sa tharollo ea 1ml ea peptide ena:
Molarity = 'Misa (0.001g) / (bophahamo ba modumo (0.001L) x 'Misa oa Molar (MW 20,000) = 50 µM

Ho fumana boima ba ho fihlella molarity e itseng:
Haeba bakeng sa tlhahlobo ea hau, o hloka tharollo ea 0.01mM e sebetsang ea peptide ka 1ml ea metsi, ebe u bala boima bo hlokahalang ka tsela e latelang:

Boima = Molarity x Volume x Molar Mass
Boima = 0.01mM x (1/1000 L) x 20,000g/mol = 0.0002g

Kahoo, o tla hloka 0.2mg/ml ea peptide ho ba le tharollo e sebetsang ea 0.01mM.


Re fana ka e le lyophilized kapa phofo e tala le tharollo ea peptide.


Ho qhibiliha ha peptide mokelikeling o fanoeng ho itšetlehile ka tatelano ea amino acid 'me hangata ho thata ho bolela esale pele.. Tabeng ea li-peptide tsa lethathamo, lintlha tsa solubility li thathamisitsoe ho database ea QC. Bakeng sa li-peptide tse tloaelehileng, ka kopo sheba karolo e nang le sehlooho se reng “Ke lokela ho hlabolla peptide ea ka joang?”.


Likahare tsa peptide ha se sesupo sa bohloeki ba peptide kaha tsena ke litekanyo tse peli tse fapaneng. Bohloeki bo khethoa ke tlhahlobo ea HPLC mme e bontša boteng / ho ba sieo ha litšila tse ling tsa peptidic (i.e. li-peptide tse fokolang).

Likahare tsa peptide tsa Net li lekanya palo ea 'nete ea peptide (peptide ea thahasello hammoho le litšila tsa peptide) e teng mohlaleng. Likahare tsa net peptide ka tloaelo li lekantsoe ka tlhahlobo ea Amino acid (AAA; ho nepahala ho fokolang empa ho hloka thepa e fokolang) kapa tlhahlobo ea mantlha (CHN; e hloka limiligrama tsa peptide empa e nepahetse haholoanyane).


MOL Liphetoho li-peptide ka kakaretso li hloekisoa ka HPLC ea ho itokisa, tshebediso 2 li-buffer tse nang le 0.1% asiti trifluoroacetic (TFA). Buffer ea pele (Buffer A) e entsoe ka 0.1 % TFA ka metsing a deionized le buffer ea bobeli (Buffer B) e entsoe ka 0.1 % TFA ka acetonitrile (ACN).

Ka kakaretso, li-peptide tse tala li qala ho qhibiliha ka tharollo ea Buffer A, palo e itseng ea Buffer B, kapa motsoako oa motsoako oa polar o kang DMSO. Haeba ho ne ho sebelisoa motsoako oa polar kapa Buffer B, tharollo ena e hlakileng e tla qala ka ho hlapolloa ka Buffer A. E 'ngoe, tharollo ea peptidic e tlhotliloeng ebe e kenngoa ho sistimi e itokisang ea HPLC. Ho sebelisa mokhoa o ntlafalitsoeng oa gradient, Joale likaroloana li bokelloa ho ipapisitsoe le lipalo tsa ho monya. Karolo e 'ngoe le e' ngoe e hlahlojoa ka HPLC ea tlhahlobo ho netefatsa hore bohloeki ba karolo e 'ngoe le e' ngoe e kopana le litlhaloso tse hlokahalang tsa bohloeki..

Likaroloana tsena tse "hloekileng" li kopantsoe hammoho 'me li na le lyophilized. Phofo "e hloekileng" ebe e lekoa bakeng sa bohloeki le boitsebiso ho netefatsa hore peptide e nepahetseng e entsoe..

Qetellong, sehlopha se ikemetseng sa QC ho AnaSpec se boetse se leka peptide ho netefatsa hore litšoaneleho tsohle tsa tlhahlobo li kopana le lintlha tse hlokahalang..


Lebitso la machaba le sebelisoang bakeng sa tatellano termini ke ka tsela e latelang:

– N-terminus: H e bolela amine ea mahala (NH2-), Ac e bolela acetyl [CH3C(O)-NH-], Pyr e bolela pyroglutamic acid
– C-terminal: OH e bolela asiti ea mahala (-COOH), NH2 e bolela amide [-CONH2]

– Liphetoho tse lehlakoreng la ketane ea li-amino acid li bontšoa ka har'a masakaneng ka mor'a amino acid e tšoanang.. Ka mohlala; phosphorylated serine = S(PO3H2) kapa epsilon-N-acetylated lysine = K(Ac)


Nakong ea tlhahiso ea peptide, li-ion tsa motsoako le litharollo tsa tlhoekiso li tlama liketane tsa lehlakore tsa amino acid tse qositsoeng tsa peptide., ho etsa matsoai. Li-ion tsena li tsejoa e le li-counterion tsa peptide mme li thusa ho leka-lekanya tefiso ea peptide.

Asiti ea Trifluoroacetic (TFA) hangata e sebelisoa ka peptide synthesis le tlhoekiso.

E etsa matsoai a trifluoroacetate ka li-amino acid tsa mantlha (mohlala, lysine, arginine, histidine) le N-terminal amine ea mahala. Ka hona, trifluoroacetate hangata ke counterion ea kamehla.

Palo e fokolang ea limolek'hule tsa TFA tse tlameletsoeng ho peptide e lekana le palo ea masala a mantlha ka tatellano e fanoeng ea peptide.. Kahoo, mesaletsa ea mantlha e teng ka har'a peptide, ho fokotsa palo ea peptide ea letlooa la phofo ea lyophilized. Ho ikarabella bakeng sa TFA kapa litaba tsa metsi tse teng sampoleng, re khothaletsa ho etsa tlhahlobo ea litaba tsa peptide bakeng sa peptide e 'ngoe le e' ngoe ea tloaelo.

Metsi le trifluoroacetic acid (TFA) ke lihlahisoa tsa ts'ebetso ea tlhoekiso. Leha ho le joalo, TFA e ka hlahisa matšoenyeho a chefo bakeng sa phela le lithuto tsa preclinical. Ka hona, Liphetoho tsa MOL li fana ka acetate e seng chefo, chloride, kapa ho fapanyetsana letsoai la ammonium.


Li-peptide tse nang le Cys, Kopana, kapa Trp e phephetsa ho kopanya le ho fumana lihlahisoa tsa boleng bo holimo. Sena se bakoa haholo ke ho se tsitse le ho ba le ts'oaetso ea oxidation ea lihlopha tsena tse sebetsang. Ho hlokahala tlhokomelo e khethehileng bakeng sa ts'ebeliso le polokelo ea li-peptide tsena ho qoba ho buloa khafetsa ha linkho.

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