Tambayoyin da ake yawan yi

Kawai zuwa hadaddun daga milligrams zuwa kilogiram

Buƙatar Quote na Musamman


How to store peptides?

Lyophilized Peptides

Don adana dogon lokaci na peptides, muna ba da shawarar adana su azaman foda mai ƙarfi. Ana iya adana peptides na lyophilized a -20 ° C ko ƙasa tare da ƙarancin lalacewa. An shawarci abokan ciniki da su sake duba peptides na musamman kowane 2 shekaru daga ranar da ingancin kula da saki.

Peptides a cikin bayani

Peptides a cikin bayani ba su da kwanciyar hankali kuma suna da saurin lalacewa. Maganin ruwa mai ruwa da ake amfani da su don narkar da peptides ya kamata su zama bakararre da tsabta.

A cikin bayani, wani adadin peptide na iya raguwa, dangane da amino acid da ke cikin jerin. An jera wasu misalai a ƙasa:
– Peptides dauke da methionine, Cystine ko ragowar tryptophan yakamata su sami iyakanceccen lokacin ajiya a cikin bayani saboda iskar oxygenation. Wadannan peptides ya kamata a narkar da su a cikin abin da ba shi da oxygen. –
Glutamine da asparagine za a iya lalata su zuwa glutamic acid da aspartic acid, bi da bi. –
Cysteine ​​​​zai iya jurewa cyclization oxidative don samar da haɗin gwiwar cysteine-cysteine ​​​​disulfide. (wanda za a iya samu a ciki ko tsakanin disulfide bond). –
Ragowar da aka caje (aspartic acid, glutamic acid, lysine, arginine, histidine) su ne hygroscopic (sha ruwa daga iska) kuma yakan haifar da danko, share mai. Wannan canji na jiki bazai shafi yanayin peptide ba. Ana ba da shawarar cewa irin waɗannan peptides su zama lyophilized don cire adadin ruwa, sannan a zubar da nitrogen don tabbatar da cewa babu tururin ruwa a cikin vial, sannan tayi saurin rufewa.

Don hana lalacewa daga maimaita daskarewa da narkewa, muna ba da shawarar cewa masu amfani kawai su narkar da adadin peptide da ake buƙata don gwajin na yanzu. Ya kamata a adana abubuwan da suka wuce kima a ≥ -20 o C har sai an buƙata.

Guji danshi.
Tun da danshi muhimmanci rage dogon lokacin da kwanciyar hankali na peptides, muna ba da shawarar daidaita peptides a cikin injin bushewa zuwa zafin jiki kafin buɗe vials. Bayan kammala rarraba peptide, sauran peptides da ke cikin bututu ya kamata a wanke su a hankali tare da nitrogen, sai a rufe kwandon, an rufe shi da fim ɗin rufewa, da kuma adana a -20 ° C.

Yadda ake narkar da peptides?

Halayen solubility na peptides daban-daban sun bambanta sosai. Ragowa kamar alanine (Ala), cysteine (Cys), isoleucine (Tare da), leucine (Leu), methionine (Haɗu), phenylalanine (Phe), da valine (Val) ƙara zuwa peptide ta hydrophobicity da solubility a cikin ruwa mafita, kuma ana ba da shawarar cewa abokin ciniki ya bi waɗannan jagororin lokacin da aka tsara peptides.

Abubuwan Solubility

Solubility na peptides ya dogara sosai akan jerin amino acid. A dabi'ance hydrophobic peptides (A, F, G, V, L, I, M, W, P tare da haɓakawa mafi girma) bukatar kwayoyin kaushi don solubilization. peptides na acidic (mafi girma ga D, E a cikin jerin peptide) suna buƙatar buffer na asali na ruwa don solubilization, yayin da asali peptides (Mafi kyawun zaɓi don K, H, da R) na buƙatar buffers mai ruwa mai acidic don narkewa.

Zaɓin Magani

Idan aka ba da iyakacin binciken ku, muna ba da shawarar yin amfani da jagororin masu zuwa don ƙayyade mafi kyawun ƙarfi don solubilizing peptides

Hydrophobic Peptides

Don sake-solubilize hydrophobic peptides, ƙara 100 µL na DMSO da sonicate har sai an samar da bayani iri ɗaya. Na gaba, ƙara buffer ɗin da kuka zaɓa don yin a 1 mg/ml bayani (mafi girman ƙwayar peptide, mafi girman adadin DMSO da ake buƙata).

Hydrophilic (Acid) Peptides

Don sake-solubilize acidic peptides, ƙara 100 µL na 1% NH 4 OH ku 1 MG na peptide da vortex. Da zarar maganin ya bayyana, ƙara buffer ɗin da kuka zaɓa don yin a 1 mg/ml bayani.

Hydrophilic (Na asali) Peptides

Don sake fasalin peptide na asali, ƙara distilled ruwa zuwa 1 MG na peptide da vortex.

Menene bambanci tsakanin net vs babban nauyin peptide?

Ma'aunin masana'antu shine isar da peptides a cikin nau'in lyophilized azaman Babban nauyi. Babban nauyin peptide shine jimlar nauyin duk abubuwan da ke cikin foda na lyophilized. Wannan ya haɗa da peptide na sha'awa, duk wani rashin lafiyan peptide, ruwa, sauran kaushi, da juriya.

Akasin haka Net nauyin peptide, lissafta daga Yanar gizo na peptide, shine nauyi na kawai peptide bangaren da ke cikin lyophilized foda. Wannan yana ba da ƙarin ingantattun ma'auni na maganin peptide na gaba. Don haka, mun ba da shawarar adadin yawan nauyin peptide don Peptide na al'ada umarni.

Net Peptide abun ciki Ana yin ma'auni ta hanyar nazarin amino acid (AAA; iyakantaccen daidaito amma yana buƙatar ƙarancin kayan abu) ko bincike na asali (CHN; yana buƙatar milligrams na peptide amma ya fi daidai). Duk hanyoyin biyu suna ba da ƙimar kashi (watau., 75% net peptide abun ciki).

Abubuwan da ke cikin Peptide na Net ya dogara da adadin abin da ake ciki *, wanda ya dogara da jerin peptide. Mahimmanci, mafi girman cajin peptide, ƙananan abun ciki na peptide

*Lura: Abubuwan da ake amfani da su don ƙididdige abun ciki na peptide na yanar gizo yana dogara ne akan counter-ion peptide. Anyi wannan don tabbatar da cewa counter-ion baya taimakawa ga adadin abun ciki na peptide. Misali, Lokacin da TFA ko acetate sune counter-ion, Ana amfani da nitrogen saboda ana samun shi a cikin peptide amma ba a cikin TFA ko acetate ba. Ana iya amfani da Carbon ko Nitrogen lokacin da chloride shine maƙarƙashiya don wannan dalili.

Lissafin Nauyin Peptide Net:

Net Peptide Quantity (jimlar peptide) - Hanyar da muke amfani da ita don odar peptide na al'ada.

Ƙirƙiri Kashi na Abubuwan Abin ciki na Peptide na Net (nau'i na goma) ta Peptide Gross Weight, don samun adadin Net Peptide Quantity (jimlar peptide).

Misali:
Domin 5 MG babban peptide / vial:
Peptide net abun ciki shine 84%.
Daidai adadin jimlar peptide = 0.84*5 mg=4.2 MG jimlar peptide

Net Peptide na Yawan Sha'awa - mafi daidai - abokin ciniki na iya amfani da wannan hanyar da kansu don lissafin peptide na sha'awa.

Haɓaka Kashi na Abubuwan Abubuwan Ciki na Net na Peptide da Babban Nauyin Peptide, da kuma Peptide Purity (ta HPLC), don lissafin ainihin adadin peptide na sha'awa da kuke da shi. Adadin peptide kashi (nau'i na goma) ana ninka ta da tsabtar peptide (nau'i na goma).

Misali:
Domin 5 MG babban peptide / vial tare da:
Peptide net abun ciki shine 84%.
Madaidaicin adadin peptide na sha'awa shine = 0.84*0.96*5 mg=4.03 mg

Wane irin sinadarai kuke amfani da shi?

Canje-canje na MOL yana amfani da sinadarai na Fmoc mai ƙarfi maimakon sunadarai na BOC. Chemistry Fmoc yana ba da damar kawar da ƙungiyoyi masu karewa ta amfani da yanayi mai laushi a lokacin elongation peptide, yayin da ilmin sunadarai na BOC na buƙatar amfani da acid mai karfi don cire ƙungiyoyi masu kariya a lokacin peptide elongation. Bugu da kari, Fmoc chemistry yana amfani da trifluoroacetic acid (TFA) don cire peptide daga guduro, yayin da BOC chemistry ke amfani da acid hydrofluoric (HF). HF ba shi da launi, sosai m, da gubar lamba.

Saboda haka, Canje-canjen MOL yana amfani da sinadarai na Fmoc kamar yadda ya fi sauƙi, m, kuma m idan aka kwatanta da BOC chemistry.

Ta yaya zan lissafta molarity na peptide na?

Molarity yana nufin haɗuwa da molar bayani, wanda shine adadin moles na solute da aka narkar dasu 1 lita na bayani, an bayyana shi azaman mol/L, ko M.

Halittu [M] = Mass / (Volume x Molar Mass); Mole = Tattaunawa (g/l) x girma (L)/MW (g/mol)

Misali:
An ba: 1MG na busassun peptide foda tare da MW: 20KDa (Molar taro na peptide ne 20 g/mol)
Don ƙayyade molarity tare da sanannun taro da ƙarar da aka sani
Don maganin 1ml na wannan peptide:
Molarity = Mass (0.001g) / (girma (0.001L) x Molar Mas (MW 20,000) = 50 µM

Don ƙayyade taro don cimma wani ƙayyadaddun molarity:
Idan don tantancewar ku, Kuna buƙatar 0.01mM maganin peptide mai aiki a cikin 1ml na ruwa, sannan a lissafta yawan adadin da ake bukata kamar haka:

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

Don haka, Kuna buƙatar 0.2mg/ml na peptide don samun maganin aiki na 0.01mM.

A cikin wane nau'i ne ake kawo peptides?

Muna isar da shi azaman lyophilized ko ɗanyen foda da maganin peptide.

Ta yaya zan san ko peptide na zai iya narkewa?

Solubility na peptide a cikin abin da aka ba da ƙarfi ya dogara ne akan jerin amino acid kuma galibi yana da wuyar tsinkaya.. A cikin yanayin catalog peptides, An jera bayanin solubility akan takaddar bayanan QC. Don peptides na al'ada, don Allah a duba sashin mai take “Yaya zan solubilize peptide na”.

Menene bambanci tsakanin abun ciki na peptide da tsarkin peptide?

Abubuwan da ke cikin peptide ba nuni bane na tsaftar peptide saboda waɗannan ma'aunai ne daban daban. Ana ƙididdige tsarki ta HPLC na nazari kuma yana nuna kasancewar/rashin sauran gurɓataccen ƙwayar cuta. (i.e. truncated peptides).

Abubuwan da ke cikin peptide na yanar gizo suna ƙayyade ainihin adadin peptide (peptide na sha'awa da kuma gurɓataccen peptide) ba a cikin samfurin. Ana auna abun ciki na peptide ta hanyar al'ada ta hanyar nazarin amino acid (AAA; iyakantaccen daidaito amma yana buƙatar ƙaramin adadin abu) ko bincike na asali (CHN; yana buƙatar milligrams na peptide amma ya fi daidai).

Ta yaya kuke tsarkake peptides gabaɗaya?

MOL Canje-canjen peptides ana tsarkake su gabaɗaya ta hanyar juyawa-lokaci na shirye-shiryen HPLC, amfani 2 buffers dauke da 0.1% trifluoroacetic acid (TFA). Buffer na farko (Buffer A) ya kunshi 0.1 % TFA a cikin ruwa mai tsafta da buffer na biyu (Buffer B) ya kunshi 0.1 % TFA a cikin acetonitrile (ACN).

Gabaɗaya, An fara narkar da ɗanyen peptides a cikin maganin ko dai Buffer A, wasu adadin Buffer B, ko kaushi mai ƙarfi kamar DMSO. Idan an yi amfani da kaushi mai ƙarfi na halitta ko Buffer B, Wannan bayyanannen bayani za a fara diluted tare da Buffer A. Na gaba, Ana tace maganin peptidic kuma an ɗora shi akan tsarin HPLC na shirye-shiryen. Yin amfani da ingantaccen tsarin gradient, sannan ana tattara ɓangarorin bisa ga karatun abin sha. Ana nazarin kowane juzu'i ta hanyar HPLC na nazari don tabbatar da cewa tsaftar kowane juzu'i ya dace da ƙayyadaddun tsafta da ake buƙata..

Waɗannan ɓangarorin “tsabta” ana haɗa su tare kuma a sanya su lyophilized. Ana gwada foda "tsabta" don duka tsarki da kuma ainihi don tabbatar da cewa an ƙera peptide daidai..

Daga karshe, ƙungiyar QC mai zaman kanta a AnaSpec ta sake gwada peptide don tabbatar da cewa duk halayen gwaji sun cika ƙayyadaddun da ake buƙata..

Ta yaya zan rubuta ƙarshen jerin peptide na?

Ƙididdigar ƙididdiga ta duniya da aka yi amfani da ita don jerin jerin kalmomi kamar haka:

– N-terminus: H yana nufin amina kyauta (NH2-), Ac yana nufin acetyl [Farashin CH3C(O)-NH-], Pyr yana nufin pyroglutamic acid
– C-terminal: OH yana nufin acid kyauta (-COOH), NH2 na nufin amide [-CONH2]

– Ana nuna gyare-gyare a kan sassan sassan amino acid a cikin bayyani bayan amino acid daidai. Misali; phosphorylated serine = S(Farashin PO3H2) ko epsilon-N-acetylated lysine = K(Ac)

Menene maganin peptide?

Lokacin samar da peptide, ions a cikin kira da mafita na tsarkakewa suna ɗaure da caje-halin sarƙoƙin gefen amino acid na peptide, kafa gishiri. Wadannan ions an san su da peptide counterions kuma suna taimakawa wajen daidaita cajin peptide.

Trifluoroacetic acid (TFA) yawanci ana amfani dashi a cikin kirar peptide da tsarkakewa.

Yana samar da salts trifluoroacetate tare da amino acid na asali (misali, lysine, arginine, histidine) da kuma N-terminal amin. Saboda haka, trifluoroacetate yawanci shine tsohuwar ƙima.

Matsakaicin adadin ƙwayoyin TFA da ke ɗaure zuwa peptide ya yi daidai da adadin abubuwan da suka rage a cikin jerin peptide da aka bayar.. Don haka, mafi asali ragowar da ke cikin peptide, ƙananan abun ciki na peptide na lyophilized foda. Don lissafin TFA ko abun ciki na ruwa da ke cikin samfurin, muna ba da shawarar yin gwajin abun ciki na peptide ga kowane peptide na al'ada.

Ruwa da kuma trifluoroacetic acid (TFA) samfurori ne na tsarin tsarkakewa. Duk da haka, TFA na iya haifar da damuwa mai guba don mai rai da kuma karatun sahihanci. Saboda haka, Canje-canje na MOL yana ba da acetate mara guba, chloride, ko ammonium gishiri musayar.

hy are peptides dauke da Cys, Haɗu, ko Trp mai wuyar haɗawa?

Peptides dauke da Cys, Haɗu, ko Trp suna da kalubale don haɗawa da samun samfurori masu tsabta. Wannan ya faru ne saboda rashin kwanciyar hankali da rashin ƙarfi ga oxidation na waɗannan ƙungiyoyi masu aiki. Ana buƙatar kulawa ta musamman don amfani da adana waɗannan peptides don guje wa sake buɗe kwalayen.

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