I-BIOSENSORS heliX eguqukayo kanye nekhithi yomenzeli yokwehlisa 1

Izici Eziyinhloko

• Ivumela ukuncishiswa kwamabhondi e-disulfide kusetshenziswa i-TCEP.
• Ihambisana namaprotheni kanye namasosha omzimba (MW> 20 kDa).
• Ukwehliswa okuqondile kwesizinda samasosha omzimba.
• Ukuhlangana ngokushesha kwama-molecule ancishisiwe kungenzeka (bheka imanuwali yomsebenzisi HK-MAL).
• Ukunciphisa ama-ligand amaningi kungenziwa ngesikhathi esifanayo.
• Kufaka phakathi ama-reagents okusabela okubili kokunciphisa ngakunye.

Incazelo Yomkhiqizo

Inombolo Ye-oda: I-RK-PA-1

Ithebula 1. Okuqukethwe kanye Nokwaziswa Kwesitoreji

Okubalulekile	Cap	Inani	Isitoreji
I-TCEP (500 mM)	Okubomvu	2 x 20µL	-20°C
Ukunciphisa i-Buffer [1]	Emhlophe	2 x 1.8 mL	-20°C
Ikholomu ye-spin yokuhlanza	Okubomvu	4 x	2-8°C
2.0 mL amashubhu wokusabela wekholomu yokuphotha yokuhlanza		4 x	RT
Iyunithi yesihlungi se-Centrifugal (10 kDa MWCO)		2 x	RT
I-tube yokuqoqa i-Centrifugation		4 x	RT

Okokusetshenziswa kocwaningo kuphela.

Lo mkhiqizo unomkhawulo wempilo yeshelufu, sicela ubone idethi yokuphelelwa yisikhathi kulebula.

KUBALULEKILE

Imikhiqizo ingathunyelwa ngamazinga okushisa ahlukene, kodwa ukugcinwa kufanele kuhambisane neziqondiso ezivezwe kuThebula. I-resin slurry kukholamu yesipina sokuhlanza iqukethe u-0.02 % sodium azide.

Izinto Ezengeziwe Ezidingekayo

Ithebula 2. Izinto Ezengeziwe

Okubalulekile	Amazwana
I-benchtop microcentrifuge	Ibanga lesivinini elidingekayo eliphakathi kuka-1,000 xg kuya ku-13,000 xg
I-Vortex
1.5 mL amashubhu okusabela

Zonke izixazululo ezidingekayo namabhafa afakiwe kukhithi.

Izinyathelo Ezi-3 Zokuncishiswa Kwe-Biomolecule Kushubhu Yokusabela

Sicela ufunde yonke inqubo ngaphambi kokuqala futhi yenza zonke izinyathelo ngaphandle kokuphazamiseka.

ICEBISO

Le phrothokholi ingenziwa kanyekanye ekuphenduleni kokuhlanganisa okuningi

I-Buffer Exchange of Protein/Antibody

1. Mmanzisa ulwelwesi lweyunithi yesihlungi se-centrifugal nge-100 µL Yokunciphisa Isibambiso.
2. Engeza cishe. 200 µg (kufika ku-500 µg) weprotheyini/amasosha omzimba kuyunithi yesihlungi ukusuka esinyathelweni 1. Uma kudingeka engeza Ukunciphisa Ibhafa kusihlungi kuze kufinyelelwe ivolumu enkulu engu-450 µL kanye ne-centrifuge ku-13,000 xg (kufika ku-14,000 xg) kokungu-5 min bese ulahla ukugeleza.
3. Engeza u-350 µL we-Reducing Buffer kanye ne-centrifuge ku-13,000 xg imizuzu emi-5 bese ulahla ukugeleza futhi.
4. Phinda isinyathelo sesi-3, kanye nesikhathi sokugcina se-centrifugation esiyimizuzu eyi-10 ukuze uthole ivolumu yokugcina ye-approx. 80µL.
5. Qoqa u-sample ngepayipi elingu-100 µL libe yishubhu entsha noma ngokubeka idivayisi yokuhlunga ibheke phansi eshubhuni elisha le-centrifugal, i-centrifuge imizuzu emi-2 ku-1000 x g.

Yehlisa nge-TCEP

1. Lungiselela isixazululo esingu-2.5 mM TCEP Ekwehliseni Ibhafa ngokwengeza u-1 µL ka-500 mM TCEP ku-199 µL Reducing Buffer.
   Lungiselela njalo ngqo ngaphambi kokusebenzisa.
2. Engeza Ukunciphisa Ibhafa ku-sample vial yenani levolumu engu-98 µL bese wengeza u-2 µL wesixazululo se-TCEP esingu-2.5 mM ukuze uthole ukugxiliswa kokugcina okungu-50 µM TCEP. Hlanganisa ukusabela ngokubhobhoza phezulu naphansi.
3. Fukamela ikhambi ku-37°C ku-400 rpm amahora angu-2 ukuqinisekisa ukuncipha okuphelele.

Ukuhlanzwa kanye Nokugxila

1. Linganisa amakholomu amabili okuhlanzwa (ikepisi elibomvu) kokuwasha okukodwa kwe-TCEP:
   a. Susa uphawu olungezansi lwekholomu bese ukhulula ikepisi (ungasusi isivalo).
   b. Faka ikholomu kushubhu yokusabela engu-2.0 mL.
   c. I-Centrifuge ku-1,500 × g imizuzu engu-1 ukususa isisombululo sokugcina.
   d. Engeza u-400 µL we-Reducing Buffer kumbhede we-resin wekholomu. I-Centrifuge ku-1,500 × g iminithi elingu-1 ukuze kukhishwe ibhafa.
   e. Phinda isinyathelo d bese ulahla ibhafa ewumphumela eshubhuni yokusabela. Ikholomu yokuphonswa kokuhlanza kufanele manje ibe sesimweni esomile.

TCEP ukugeza

a. Beka amakholomu esinyathelweni sangaphambilini kumashubhu okusabela amasha angu-1.5 mL.
b. Susa isivalo sekholomu yesipina esingu-1 bese uthela kahle u-sample phezulu kombhede we-resin.
c. I-Centrifuge ku-1,500 xg imizuzu emi-2 ukuqoqa i-sample (ukugeleza). Lahla ikholomu ye-spin yokuhlanza ngemva kokusetshenziswa.
d. Susa isivalo sekholomu yesi-2 bese uthela u-sample ukusuka esinyathelweni sangaphambili ukuya embhedeni we-resin.
e. I-Centrifuge ku-1,500 xg imizuzu emi-2 ukuqoqa i-sample (ukugeleza). Lahla ikholomu ye-spin yokuhlanza ngemva kokusetshenziswa.
f. I-Thiol coupling ngokuya ngemanuwali yomsebenzisi (HK-MAL-1 noma i-HK-MAL-2) ye-s encishisiweample ingenziwa.

Ulwazi Olwengeziwe

I-Buffer Exchange and Concentration with Centrifugal Filter Units 

1. Thatha iyunithi yokuhlunga eyodwa ye-centrifugal, engeza ivolumu efanelekile yebhafa kudivayisi yokuhlunga, bese uyivala.
2. Beka idivayisi yokuhlunga efakwe i-capped ku-rotor ye-centrifuge, uqondanise ibhande le-cap liye phakathi nendawo ye-rotor; ukumelana nedivayisi efanayo.
3. Spina idivayisi ku-13,000 xg (noma 14,000 xg) ngesikhathi esinikeziwe.
4. Susa ukugeleza bese uphinda izinyathelo 1-3.
5. Susa idivayisi ehlanganisiwe ku-centrifuge futhi uhlukanise idivayisi yokuhlunga neshubhu le-microcentrifuge.
6. Ukuze ubuyisele i-conjugate, beka idivayisi yokuhlunga ibheke phansi eshubhuni elihlanzekile le-centrifugal, uqondanise ikepisi elivulekile liye phakathi nendawo yerotha; ukumelana nedivayisi efanayo. Spin imizuzu emi-2 ku-1,000 xg ukuze uthumele i-sample kusuka kudivayisi kuya tube.
   

Izinombolo Ze-oda Eziwusizo

Ithebula 3. Izinombolo ze-oda

Igama Lomkhiqizo	Inani	I-oda No
I-heliX® Thiol coupling kit 1	5 x	I-HK-MAL-1
I-heliX® Thiol coupling kit 2	5 x	I-HK-MAL-2
Iyunithi yesihlungi se-Centrifugal (3 kDa MWCO)	Ama-5 ama-pcs.	CF-003-5
Iyunithi yesihlungi se-Centrifugal (10 kDa MWCO)	Ama-5 ama-pcs.	CF-010-5
10x ibhafa A [2]	50 mL (ikhiqiza 500 mL)	BU-P-150-10
5x ibhafa B [3]	50 mL (ikhiqiza 250 mL)	BU-P-1000-5

Oxhumana naye

I-Dynamic Biosensors GmbH	Inkampani Dynamic Biosensors, Inc
I-Perchtinger Str. 8/10	300 Trade Center, Suite 1400
81379 eMunich	I-Woburn, i-MA 01801
EJalimane	USA

Oda Ulwazi order@dynamic-biosensors.com
Ukusekela kwezobuchwepheshe support@dynamic-biosensors.com

1. Yehlisa Ibhafa: 100 mM Na2HPO4/NaH2PO4, 300 mM NaCl, 5 mM EDTA, pH 7.4
2. Buffer A: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.2
3. I-Buffer B: 50 mM Na2HPO4/NaH2PO4, 1 M NaCl, pH 7.2

USEKELO LWEKHASIMENDE

www.dynamic-biosensors.com
Izinsimbi nama-chips akhiwe futhi akhiqizwa eJalimane.
©2024 Dynamic Biosensors GmbH | Dynamic Biosensors, Inc. Wonke amalungelo agodliwe.

Amadokhumenti / Izinsiza

I-BIOSENSORS heliX eguqukayo kanye nekhithi yomenzeli yokwehlisa 1 [pdf] Imaniwali yosebenzisayo I-RK-PA-1, i-heliX plus Reducing Agent Kit 1, i-heliX plus, Ikhithi yomenzeli 1 yokunciphisa, Ikhithi yomenzeli 1, Ikhithi engu-1

Izithenjwa

• Imaniwali yosebenzisayo