dynamic BIOSENSORS heliX gbakwunyere Mbelata Agent Kit 1

Atụmatụ igodo

• Na-enye ohere ibelata nkekọ disulfide site na iji TCEP.
• Dakọtara na protein na ọgwụ mgbochi (MW> 20 kDa).
• Mbelata ọgwụ mgbochi ọrịa na saịtị.
• Ngwa ngwa njikọ nke ụmụ irighiri ihe dị ala ga-ekwe omume (tụ aka na ntuziaka onye ọrụ HK-MAL).
• Enwere ike ime mbelata ọtụtụ ligands n'otu oge.
• Gụnyere reagents maka mmeghachi omume mbelata mmadụ abụọ.

Nkọwa ngwaahịa

Nọmba ịtụ ahịa: RK-PA-1

Tebụl 1. Ọdịnaya na Ozi Nchekwa

Ihe onwunwe	Okpu	Ọnụ ego	Nchekwa
TCEP (500 mm)	Uhie	2 x 20 µL	-20°C
Na-ebelata ihe nchekwa [1]	Ọcha	2 x1.8 ml	-20°C
Kọlụm atụ ogho dị ọcha	Uhie	4 x	2-8 Celsius C
2.0 ml mmeghachi omume tubes maka ịdị ọcha spin kọlụm		4 x	RT
Otu nzacha Centrifugal (10 kDa MWCO)		2 x	RT
Centrifugation tube mkpokọta		4 x	RT

Maka iji naanị nyocha.

Ngwaahịa a nwere ndụ nchekwa nwere oke, biko hụ ụbọchị ngwụcha na label.

IHE dị mkpa

Enwere ike ibupu ngwaahịa na okpomọkụ dị iche iche, mana nchekwa kwesịrị ịgbaso ntuziaka ndị akọwapụtara na tebụl. Ngwunye resin dị na kọlụm ịhịa aka n'ahụ nwere 0.02% sodium azide.

Ihe ndị ọzọ achọrọ

Tebụl 2. Ihe ndị ọzọ

Ihe onwunwe	Okwu
Benchtop microcentrifuge	Oke ọsọ achọrọ dị n'etiti 1,000 xg ruo 13,000 xg
Vortex
1.5 ml mmeghachi omume tubes

A na-etinye ihe ngwọta niile dị mkpa na ihe nchekwa na ngwa ahụ.

Nzọụkwụ 3 Mbelata Biomolecule na tube mmeghachi omume

Biko gụọ protocol niile tupu ịmalite na mee usoro niile na-enweghị nkwụsị.

NDỤMỌDỤ

Enwere ike ịme ụkpụrụ a n'otu oge maka ọtụtụ mmeghachi omume njikọta

Mgbanwe nke Protein/Antibody

1. Weta akpụkpọ ahụ nzacha centrifugal na 100 µL na-ebelata ihe nchekwa.
2. Tinye ihe ruru. 200 µg (ruo 500 µg) nke protein / antibody na nzacha nzacha site na nzọụkwụ 1. Ọ bụrụ na ọ dị mkpa gbakwunye Reducing Buffer na nyo ruo mgbe oke olu 450 µL ruru na centrifuge na 13,000 xg (ruo 14,000 xg) maka 5. min ma tụfuo eruba-site.
3. Tinye 350 µL nke Mbelata ihe nchekwa na centrifuge na 13,000 xg maka nkeji 5 wee tụfuo erute-site ọzọ.
4. Tinyegharịa nzọụkwụ 3, otu oge ọzọ na oge centrifugation ikpeazụ nke 10 min iji nweta olu ikpeazụ nke ihe ruru. 80 µL.
5. Anakọta sampma ọ bụ jiri 100 µL pipette n'ime tube ọhụrụ ma ọ bụ site n'itinye ngwaọrụ nzacha ihu n'elu na tube centrifugal ọhụrụ, centrifuge maka 2 min na 1000 x g.

Mbelata na TCEP

1. Kwadebe ihe ngwọta 2.5 mM TCEP na Mbelata ihe nchekwa site na ịgbakwunye 1 µL nke 500 mM TCEP n'ime 199 µL Mbelata ihe nchekwa.
   Kwadebe mgbe niile ozugbo tupu ojiji.
2. Tinye Mbelata ihe nchekwa na sample vial maka mkpokọta olu nke 98 µL wee gbakwunye 2 µL nke 2.5 mM TCEP ngwọta iji nweta ntinye ikpeazụ nke 50 µM TCEP. Gwakọta mmeghachi omume site na pipetting elu na ala.
3. Tinye ihe ngwọta na 37 ° C na 400 rpm maka 2 h iji hụ na mbelata zuru oke.

Ịdị ọcha na itinye uche

1. Hazie kọlụm ntugharị ọcha abụọ (okpu uhie) maka otu TCEP sachaa:
   a. Wepu akara ala kọlụm ma hapụ okpu (ewepụla okpu).
   b. Debe kọlụm na tube mmeghachi omume 2.0 ml.
   c. Centrifuge na 1,500 × g maka 1 min iji wepụ ihe ngwọta nchekwa.
   d. Tinye 400 µL nke Mbelata ihe nchekwa na akwa resin kọlụm. Centrifuge na 1,500 × g maka nkeji 1 iji wepụ ihe nchekwa.
   e. Tinyegharịa nzọụkwụ d wee tụfuo ihe nchekwa na-esi na tube mmeghachi omume pụta. Kọlụm ịhịa aka n'ahụ kwesịrị ịdị ọcha ugbu a.

TCEP ịsacha

a. Tinye ogidi ndị ahụ site na nzọụkwụ gara aga na tubes mmeghachi omume 1.5 ml ọhụrụ.
b. Wepu okpu nke ntugharị kọlụm nọmba 1 wee wụsa nke ọma sample n'elu akwa resin.
c. Centrifuge na 1,500 xg maka nkeji 2 iji nakọta sample (njedebe-site). Tụfuo kọlụm ntugharị ọcha mgbe ejiri ya.
d. Wepu okpu nke ntugharị kọlụm nọmba 2 wee wụsa sample site na nzọụkwụ gara aga na akwa resin.
e. Centrifuge na 1,500 xg maka nkeji 2 iji nakọta sample (njedebe-site). Tụfuo kọlụm ntugharị ọcha mgbe ejiri ya.
f. Njikọ Thiol dị ka akwụkwọ ntuziaka onye ọrụ (HK-MAL-1 ma ọ bụ HK-MAL-2) nke s belataraampenwere ike ịrụ ya.

Ozi Mgbakwunye

Mgbanwe nchekwa na itinye uche na nkeji nzacha Centrifugal 

1. Were otu ihe nzacha centrifugal, gbakwunye ụda nchekwa kwesịrị ekwesị na ngwaọrụ nzacha, ma kpuchie ya.
2. Tinye ihe nzacha mkpuchi mkpuchi n'ime rotor centrifuge, na-edozi eriri okpu n'etiti etiti rotor; counterbalance na ngwaọrụ yiri ya.
3. Tụgharịa ngwaọrụ ahụ na 13,000 xg (ma ọ bụ 14,000 xg) maka oge enyere.
4. Wepu ihe na-asọ asọ ma kwugharịa usoro 1-3.
5. Wepu ngwaọrụ agbakọtara na centrifuge wee kewaa ngwaọrụ nzacha na tube microcentrifuge.
6. Iji nwetaghachi conjugate ahụ, tinye ngwaọrụ nzacha ihu na ala na tube centrifugal dị ọcha, na-edozi okpu mepere emepe n'etiti etiti rotor; counterbalance na ngwaọrụ yiri ya. Tụgharịa maka nkeji 2 na 1,000 xg iji nyefee sample site na ngwaọrụ na tube.
   

Ọnụọgụ ọnụ ahịa bara uru

Tebụl 3. Nye nọmba

Aha ngwaahịa	Ọnụ ego	Iwu Mba
ngwa njikọ nke heliX® Thiol 1	5 x	HK-MAL-1
ngwa njikọ nke heliX® Thiol 2	5 x	HK-MAL-2
Otu nzacha Centrifugal (3 kDa MWCO)	5 PC.	CF-003-5
Otu nzacha Centrifugal (10 kDa MWCO)	5 PC.	CF-010-5
10x nchekwa A [2]	50 ml (ihe dị ka 500 ml)	BU-P-150-10
5x nchekwa B [3]	50 ml (ihe dị ka 250 ml)	BU-P-1000-5

Kpọtụrụ

Dynamic Biosensors GmbH	Ọnọdụ nke Dynamic Biosensors, Inc
Perchtinger Str. 8/10	300 Trade Center, Suite 1400
81379 Munich	Woburn, MA 01801
Germany	USA

Ozi ịtụ order@dynamic-biosensors.com
Nkwado ndị teknuzu support@dynamic-biosensors.com

1. Na-ebelata ihe nchekwa: 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. Buffer B: 50 mM Na2HPO4/NaH2PO4, 1M NaCl, pH 7.2

Nkwado ndị ahịa

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Akwụkwọ / akụrụngwa

dynamic BIOSENSORS heliX gbakwunyere Mbelata Agent Kit 1 [pdf] Akwụkwọ ntuziaka onye ọrụ RK-PA-1, heliX gbakwunyere Mbelata Agent Kit 1, heliX gbakwunyere, Mbelata Agent Kit 1, Agent Kit 1, Kit 1

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