Ungayisebenzisa kanjani i-glue yokuhlukanisa?– Isiqephu 1

Ungayisebenzisa kanjani i-glue yokuhlukanisa?

Ukudluliswa kwe-Western blot membrane

Ngaphansi kwesenzo samanje, iphrotheni idluliswa isuka kujeli iye kumthwali oqinile (imembrane).

Ukukhethwa kwe-Membrane: izinto ezisetshenziswa kakhulu zesigaba esiqinile ekuphrinteni zihlanganisa ulwelwesi lwe-NC, i-DBM, i-DDT, ulwelwesi lwenayiloni, i-PVDF, njll.Kunezincazelo ezimbili: immobilon-p (0.45um) kanye ne-immobilon PSQ (0.2um ye-MW <20kDa).

Indlela ye-semi dry

Okusho ukuthi, inhlanganisela ye-jeli interlayer ibekwe phakathi kwephepha lokuhlunga elinebhafa yokudlulisa, futhi okwamanje kuqhutshwa ngebhafa ekhangisiwe ephepheni lokuhlunga ukuze kuzuzwe umphumela wokudlulisa.Ngenxa yokuthi izenzo zamanje ngokuqondile ku-glue yefilimu, izimo zokudlulisa zinzima kakhulu, kodwa isikhathi sokudlulisa sifushane futhi ukusebenza kahle kuphezulu.

(1) ukukhethwa kwezimo zokuhlola

Okwamanje yi-1ma-2ma / cm2, futhi ngokuvamile sisebenzisa i-100mA / membrane.Isikhathi sokudlulisa singakhethwa ngokuhambisana nobukhulu be-molecule ye-protein ehlosiwe kanye nokuhlushwa kwe-gel, futhi ingalungiswa ngokufanele ngokuvumelana nesimo sangempela.

Khomba usayizi wamangqamuzana amaprotheni (kDa), ukugxila kwejeli nesikhathi sokudlulisa

80---140 8% 1.5-2.0

25---80 10% 1.5

15—40 12% 0.75

<20 15% 0.5

(2) ukusebenza kokuhlola

(1) Ukulungiswa kwephepha lokuhlunga kanye nolwelwesi (ukulungiswa kuzoqalwa imizuzu engama-20 ngaphambi kwe-electrophoresis).

I. Hlola ukuthi akhona yini amabhafa okudlulisa anele, angalungiswanga ngokushesha.

B. Hlola ukuthi ingabe likhona yini iphepha lokuhlunga kanye nolwelwesi olunosayizi ofanele.

C. Gxilisa ulwelwesi ku-methanol cishe imizuzu engu-1-2.Bese idluliselwa kusigcinalwazi sokudlulisa.

D. Cwilisa iphepha lesihlungi elinamathelayo elifanele kanye nephepha lokuhlunga ulwelwesi kubhafa yokudlulisa ngokulandelana.

Kufanele ulwelwesi lwe-PDVF lucwilise isikhathi esingakanani ku-methanol lapho lungena kubhafa yokudlulisa?

I-PVDF i-hydrophobic, futhi kunzima ukucwiliswa ku-buffer yokudlulisa.Ngemuva kokwelashwa kwe-methanol, kulula ukucwilisa.Ukwelashwa kwangaphambili kwe-PVDF kuwukucwilisa nokuyivula nge-methanol, bese igeza kabili ngamanzi acwecwe ngemva kokucwiliswa kahle.Injongo yokusebenzisa amabhamuza e-methanol iwukwenza kusebenze amaqembu ashajwe kahle kulwelwesi lwe-PVDF nokwenza kube lula ukuhlanganisa amaprotheni ashajiwe kabi.

Abantu bavame ukuyisebenzisa imizuzu engu-5-20.Ingacwiliswa ngesikhathi esifanayo nokuthatha iglue, futhi kulinganiselwa ukuthi kuzothatha cishe imizuzu emi-5.Umphumela awumubi.Esikhathini esedlule, abanye abangani bewebhu bathumele ukuthi isikhathi sokucubungula asinandaba kangako, kodwa ukhiye kwakuyikhwalithi yefilimu.Yiqiniso.Uma nje i-membrane igcwele ngokuphelele, kufanele ilungile.

Incazelo ye-PVDF ye-Hybond ifundeka kanje: "ngaphambi kokumanzisa ulwelwesi ngo-100% metanol (imizuzwana eyi-10)".Ukuqonda kwami ​​​​ukuthi kulungile ukucwilisa okungenani imizuzwana eyi-10.Eqinisweni, KULUNGILE ukucwilisa imizuzwana eyi-10 noma imizuzu eyi-10.

(2) Ukudlulisa

A. Beka iphepha lokuhlunga elingezansi ethuluzini lokudlulisa likagesi.Ngokuvamile kusetshenziswa izingqimba ezintathu.

B. Beka ulwelwesi ephepheni lokuhlunga ngokumelene nolwelwesi, qaphela ukuthi ungabi namabhamuza phakathi kolwelwesi nephepha lokuhlunga, bese uthela isivimbeli sokudlulisa kulwelwesi ukuze ugcine ulwelwesi lumanzi.

C. Hlanza i-glue, susa ijeli yesitaki bese uyihambisa ngokucophelela kwifilimu.

D. Sika ikhoneni eliphezulu kwesokunxele lefilimu bese uphawula indawo yeglue efilimu ngepensela.

E. Mboza ucezu lwephepha lokuhlunga ngokumelene neglue ku-glue.Thela isigcinalwazi sokudlulisa bese ubeka izingcezu ezimbili zephepha lokuhlunga.

F. Faka ithuluzi lokudlulisa ugesi bese ukhetha okwamanje nesikhathi esidingekayo ngokwezidingo.

G. Phakathi nenqubo yokudlulisa, ukuguqulwa kwamandla kagesi kuzobhekwa noma nini, futhi noma yikuphi okungavamile kuzolungiswa ngesikhathi.