Litaba pata
1 BIOSENSORS Y-Structure Amine Coupling Kit 1
2 Likarolo tsa Bohlokoa
3 Mosebetsi o Felletseview
4 Tlhaloso ea Sehlahisoa
5 Lisebelisuoa Tse Eketsehileng Tse Hlokehang
6 Lintlha tsa Bohlokoa Kakaretso
7 Khokahano ea Mehato e 3 ea Biomolecule ho Ligand strand 1
8 Tlhahisoleseling e 'Ngoe
9 Letlapa la Tšebelisano
10 Linomoro tsa Odara tse Molemo
11 Ikopanye
12 Litokomane / Lisebelisoa
12.1 Litšupiso

dynamic-BIOSENSOR-logo

BIOSENSORS Y-Structure Amine Coupling Kit 1

dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-product

Likarolo tsa Bohlokoa

  • Allows for coupling of biomolecules with primary amines (e.g. NH2-terminus, lysines) to the new Y-structure Ligand strand 1 – Red.
  • Khemistri e tloaelehileng e bonolo (k'hemistri ea NHS).
  • E lumellana le heliX® Adapter Chip.
  • E lumellana le proFIRE® tlhoekiso bakeng sa li-conjugate tsa ligand-DNA tse hloekileng (> 5 kDa).
  • Ho kopanya li-ligands tse ngata ho ka etsoa ka nako e le 'ngoe.
  • Lihlahisoa> 95 % pure ligand-DNA conjugate le mosebelisi boleng ba boleng ba sehlahisoa sa ho qetela.
  • Includes reagents for three individual conjugation reactions (approx. 10-50 regenerations each; up to max. 500).
  • E lumellana le mokhoa o ikemetseng oa ho nchafatsa maemo.

Mosebetsi o Felletseview

3-Mehato ea ho Kopanya Mosebetsi oa Ts'ebetso

BIOSENSORS Y-Structure Amine Coupling Kit 1

Nako ea nako: Matsoho ka nako < 1 h | Incubation ~ 2 h | Kakaretso ~ 3 h

Tlhaloso ea Sehlahisoa

Nomoro ea Odara: HK-NYS-NHS-1
Lethathamo la 1. Litaba le Boitsebiso ba Polokelo

Lintho tse bonahalang Cap Chelete Polokelo
Y-structure Ligand strand 1 NHS Boputsoa 3 x -20°C
Buffer A [1] Tse pepeneneng 1 x 1.8 mL -20°C
Buffer C [2] Tse pepeneneng 3 x 1.8 mL -20°C
Buffer PE40 [3] Tse pepeneneng 3 x 1.5 mL -20°C
ddH2O Tse pepeneneng 1.5 mL -20°C
Crosslinker Brown 3 x -20°C
Hloekiso spin kholomo Khubedu 6 x 2-8°C
2.0 mL reaction tubes for purification spin column   6 x RT
Sefate sa Centrifugal (3 kDa MWCO)[4]   3 x RT
Tube ea pokello ea centrifugation   3 x RT

Bakeng sa tšebeliso ea lipatlisiso feela.
Sehlahisoa sena se na le nako ea nako ea shelofo, ka kopo, sheba letsatsi la ho felloa ke nako ho label.

BOHLOKOA
Lihlahisoa li ka romelloa ka lithempereichara tse fapaneng, empa polokelo e lokela ho latela tataiso e boletsoeng ho Lethathamo.
Thepa e na le li-reagents tse lekaneng bakeng sa likhokahano tse hlano tsa hoo e ka bang 50-200 µg ea biomolecule ka 'ngoe.
Resin slurry e kholomong ea ho hloekisa e na le 0.02% sodium azide.

Lisebelisuoa Tse Eketsehileng Tse Hlokehang

Lethathamo la 2. Lisebelisoa tse Eketsehileng

Lintho tse bonahalang Maikutlo
Benchtop microcentrifuge Lebelo le hlokahalang la lipakeng tsa 1,000 xg ho isa ho 13,000 xg
Pherekano  
1.5 ml ea methapo ea methapo  
UV-Vis Spectrophotometer (mohlala, Nanodrop) Bakeng sa boikemisetso ba Ligand strand 1 mohopolo oa conjugate

Litharollo tsohle tse hlokahalang le li-buffers li kenyellelitsoe ka har'a kit.

Lintlha tsa Bohlokoa Kakaretso

  • a. The lyophilized Ligand strand e ka 'na ea se ke ea fumanoa kamehla ka tlaase ho tube; e ne e ka lula leboteng la tube kapa ka har'a cap cap. Ka kopo kamehla hlahloba boteng ba lyophilized Ligand strand, e khetholloang ka ponahalo ea eona e hlakileng ea pellet (ho ka 'na ha hlokahala hore u tlose label ea tube ho e bona). Haeba e le sieo tlase, ka kopo, kenya tube ka lebelo le holimo metsotso e 'maloa pele u qhala DNA ka har'a buffer. Ntle le moo, beha ntlha ea pipette ea hau haufi le pellet ea DNA 'me u fane ka buffer ka ho toba ho eona; DNA e tla qhibiliha ka potlako.
  • b. Crosslinker e tla amahanngoa le lihlopha tsa mantlha tsa amine (-NH2) tsa ligand. Li-amine tsa mantlha li teng N-terminus ea ketane ka 'ngoe ea polypeptide le ka har'a ketane e ka thoko ea masalla a lysine amino acid.
  • c. Qoba ho sebelisa li-buffers tse nang le li-amine tsa mantlha (ke hore, Tris, Glycine) nakong ea ts'ebetso ea ho kopanya (Ka kopo, sheba karolo ea Letlapa la Tšebelisano).
  • d. Ho fihla ho 1 mM ea Dithiothreitol (DTT) e ka sebelisoa nakong ea ho kopanya. Qoba ho sebelisa 2-Mercaptoethanol kapa lisebelisoa life kapa life tse fokotsang thiol nakong ea ts'ebetso ea ho kopanya. Haeba ho hlokahala ho fokotsa ho hlokahala, TCEP e khothaletsoa ho fihla ho 1 mmM.
  • e. Qoba ho sebelisa liprotheine tse hloekisitsoeng ka mokhoa o itsengamples kapa protheine samptse nang le bajari (mohlala, BSA).
  • f. Ho netefatsa lihlahisoa tse phahameng ka ho fetisisa tsa karabelo, ligand e lokela ho qhibiliha Buffer C. Buffer exchange e buelloa pele ho ts'ebetso ea conjugation.
  • g. Pele o qala, ka bokhuts'oane centrifuge li-tubes tsohle ka likatiba tse putsoa, ​​tse sootho le tse bonaletsang ho etsa bonnete ba hore lisebelisoa tsohle li ka tlase ho li-tubes.
  • h. Bakeng sa limolek'hule tse nang le boima ba limolek'hule tse pota-potileng kapa tse ka tlaase ho 5 kDa, ho hlokahala tlhokomelo e eketsehileng nakong ea ho hloekisa. Limolek'hule tse nyane le li-peptide tse ling li kanna tsa se hloekisoe hantle ho sebelisoa kholomo e fanoeng ea chromatographic. Ho fumana lintlha tse ling, ka kopo imeile support.dbs@bruker.com.
  • ke. Haeba pI ea protheine e le <6, pH kit e tlaase bakeng sa conjugation (Taelo ea Nomoro: HK-NHS-3) e khothalletsoa. Ho fumana lintlha tse ling, ka kopo imeile support.dbs@bruker.com.

Khokahano ea Mehato e 3 ea Biomolecule ho Ligand strand 1

Ka kopo bala protocol eohle pele u qala le ho etsa mehato eohle ntle le tšitiso.

KELETSO

Protocol ena e ka etsoa ka nako e le 'ngoe bakeng sa liketso tse ngata tsa ho kopanya.
Qoba ho sebelisa liprotheine tse hloekisitsoeng ka mokhoa o itsengamples kapa protheine samptse nang le bajari (mohlala, BSA).
Pele o qala lumella crosslinker ho fihlela mocheso oa kamore pele o sebelisoa.
Ho tloha joale ho ea pele, Y-structure Ligand strand 1 NHS e tla bitsoa Ligand strand 1.

Phetoho ea Nanolever

  1. Dissolve Ligand strand 1 in 40 µL Buffer A prior to use, vortex until all solids are completely dissolved and briefly spin down.
  2. Dissolve the crosslinker (brown cap) by adding 100 µL ddH2O, vortex until all solids are completely dissolved and briefly spin down. IMPORTANT: Always use fresh compound.
  3. Add 10 µL of the freshly prepared linker solution to one Ligand strand 1 aliquot. Discard the remaining linker solution from step 2.
  4. Tlosa li-reactants bakeng sa metsotsoana e 10, phunya fatše 'me u e kenye metsotso e 20 mocheso oa kamore.
    BOHLOKOA U se ke ua feta nako ea ho incubation kapa lihlahisoa tsa karabelo li tla fokotseha.
  5. Ho sa le joalo, kopanya litšiea tse peli tsa tlhoekiso (sekoaelo se sefubelu) bakeng sa karabelo e le 'ngoe ea ho kopanya:
    • a. Tlosa tiiso e ka tlase ea kholomo 'me u lokolle cap (u se ke ua tlosa cap).
    • b. Beha kholomo ka har'a tube ea 2.0 mL ea reaction.
    • c. Centrifuge at 1,500 x g for 1 minute to remove the storage solution.
    • d. Add 400 µL of Buffer C to the column’s resin bed. Centrifuge at 1,500 x g for 1 minute to remove buffer.
    • e. Pheta mohato oa d 'me u lahle buffer e hlahisoang ho tsoa ho tšupu ea karabelo. Kholomo ea ho hloekisa e lokela ho ba boemong bo omileng.
  6. Sample loading
    • a. Beha litšiea ho tloha mohato oa 5 ho li-tubes tse ncha tsa 1.5 mL.
    • b. Tlosa kepisi ea spin kholomo ea nomoro ea 1 'me u sebelise sample ho tloha mohato oa 4 ho ea holimo ho bethe ea resin.
    • c. Centrifuge at 1,500 x g for 2 minutes to collect the sample (phallo-through). Lahla karolo ea Purification spin ka mor'a tšebeliso.
    • d. Tlosa kepisi ea spin kholomo ea nomoro ea 2 'me u sebelise sample ho tloha mohato oa c ho ea betheng ea resin.
    • e. Centrifuge at 1,500 x g for 2 minutes to collect the sample (phallo-through). Lahla karolo ea Purification spin ka mor'a tšebeliso.

Khokahano ea Ligand

  1. Kenya hoo e ka bang. 100 µg (ho fihlela ho boholo ba 200 µg) ea ligand (concentration approx. 0.5 - 50 mg/mL) ho ea s.ample ho tloha mohato oa 6. Bakeng sa maemo a nepahetseng sebelisa boholo ba hoo e ka bang. 50 μL.
    EXAMPLE: Fetola motsoako oa protheine ho 2 mg/mL 'me u sebelise 50 µL bakeng sa ho kopanya.
    BOHLOKOA Etsa bonnete ba hore polokelo ea ligand ha e na li-amine tsa mantlha, mohlala, li-buffers tsa Tris, glycine (ka kopo, sheba Lintlha tsa Bohlokoa).
  2. Kopanya karabelo ka ho kenya liphaephe holimo le tlase 'me u e lumelle hore e arabele mocheso oa kamore bonyane hora e le 1.
    BOHLOKOA U se ke ua vortex. Haeba ho hlokahala, karabelo e ka etsoa ka 4 ° C ka nako e telele ea karabelo (mohlala, bosiu bo le bong).

proFIRE® Tlhoekiso

  1. Etsa tlhoekiso u sebelisa mokhoa o nepahetseng oa ho sebetsa oa proFIRE® (ka kopo, sheba Buka ea Mosebelisi ea proFIRE®). Ka kopo etsa bonnete ba hore sampbophahamo ba eona ke 160 μL.
    1. a. Haeba molumo o le ka tlase ho 160 µL, tlatsa molumo o sieo ka Buffer A.
      b. Haeba molumo o feta 160 µL, ka kopo etsa 160 µL e eketsehileng ho fihlela sample chesitsoe.
    2. Sebelisa Data Viewsoftware ea proFIRE® ho tseba hore na ke likarolo life tse nang le conjugate e hloekileng.
      Mohlankanaample chromatogram e bontšoa karolong ea Boitsebiso bo Eketsehileng: proFIRE® tlhoekiso ea Ligand strand 1 conjugate.
    3. Tlosa likaroloana tse khothaletsoang ho mokelli oa likaroloana.

KELETSO

Se ke oa boloka Ligand strand 1 conjugate nako e telele ho proFIRE® e sebetsang buffer. Tsoela pele hang-hang ka phapanyetsano ea buffer.

Buffer Exchange

  1. Add 500 µL of the first proFIRE® fraction containing the Ligand strand 1 conjugate to the centrifugal filter unit. Centrifuge at 13,000 x g (up to 14,000 x g) for 10 minutes and discard the flow-through.
  2. Eketsa likaroloana tse setseng ho yuniti e tšoanang ea sefe 'me u phete mohato oa centrifugation e le hore u bokelle likarolo tsohleampka har'a tube e le 'ngoe. (Ka kopo, sheba Boitsebiso bo eketsehileng: Buffer Exchange le Concentration le Centrifugal Filter Units).
  3. Kenya 350 µL ea PE40 (kapa TE40, HE40) buffer le centrifuge ho 13,000 xg metsotso e 10. Lahla phallo-ho feta.
    BOHLOKOA Haeba protheine e sa tsitsa ho PE40 (kapa TE40, HE40), ka kopo, hlahloba ts'ebetso ea buffer le letlapa la ts'ebetso ea switchSENSE®.
  4. Kenya 350 µL ea PE40 (kapa TE40, HE40) buffer le centrifuge ho 13,000 xg metsotso e 15. Lahla phallo-ho feta.
  5. To recover the Ligand strand 1 conjugate, place the centrifugal filter unit upside down in a new centrifugal collection tube (provided in the kit).
    Spin ho 1,000 xg bakeng sa metsotso e 2 ho fetisetsa sample ho tube.

Aliquots le Storage

  1. Measure the absorbance of the Ligand strand 1 conjugate at 260 nm dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-2 ho UV-Vis Spectrophotometer (mohlala, Nanodrop).
  2. Determine the concentration of the Ligand strand 1 conjugate (c1) by inserting dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-2into the following equation:dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-3where d is the path length (usually equal to 1 cm; however, please check the UV-Vis Spectrophotometer user manual) and  dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-7is the extinction coefficient of the DNA at 260 nm, equal to 272,000.
  3. Bakeng sa tharollo e seng e loketse ho sebelisoa bakeng sa ts'ebetso ea biochip, ka kopo fetola mohopolo ho 500 nM (kapa ho fihla ho 1 µM) ka PE40 (kapa TE40, HE40) buffer (ho kenyeletsoa ho fihla ho 10 % glycerol, ha ho hlokahala) 'me u lokise li-aliquots tse 20 µL. .
  4. Boloka pakeng tsa -86 °C le 8 °C, ha u batla.
    Ho tsitsa ha tharollo ho amana le botsitso ba molek'hule ea ligand.
    BOHLOKOA Pele ho tekanyo ea ho sebelisana ha switchSENSE®, ka kopo eketsa khoele e nepahetseng ea adaptara tharollong ea conjugate.

Tlhahisoleseling e 'Ngoe

proFIRE® tlhoekiso ea Ligand strand 1 conjugate

  1. Ho netefatsa liphetho tse ntle ho tsoa ho tekanyo, ha ho na Ligand strand 1 ea mahala e lokelang ho ba teng ho chip. Ka hona, li-conjugate tsa Ligand 1 tse tala li tlameha ho hloekisoa ka ion exchange chromatography pele ho tekanyo. Mohato ona oa ho laola boleng o u fa lintlha tse ling tsa bohlokoa mabapi le sample bohloeki.
  2. Re khothaletsa ho sebelisa proFIRE® sistimi e nang le kholomo ea phapanyetsano ea ion, Buffer A [1] le Buffer B [5], tse nang le sebopeho se ts'oanang, empa letsoai le fapaneng, le lumellang karohano ea tlhoro.
    BOHLOKOA Bakeng sa Y-structure Ligand strand 1 - NHS kit, ka kopo kenya 24 e le bolelele ba DNA (Mofuta oa 1) ha u qala lenaneo la tlhoekiso.
    In Figure 1 a typical proFIRE® chromatogram of a Ligand strand 1 conjugate purification is depicted, where the peak of the protein-DNA conjugate is separated from the free protein (left) and the free DNA (right).
    BOHLOKOA: The proFIRE® system owns a tailored software for automatic recognition and quantitation of DNA conjugates.
  3. Kamora ho hloekisoa, bokella likaroloana tsa li-conjugate tsa Ligand strand 1 (setšoantšo sa 1: likaroloana 8-10), tsepamisa mohopolo, 'me u fapanyetsane buffer le buffer ea khetho ea hau u sebelisa yuniti ea filthara ea Centrifugal, joalo ka ha ho hlalositsoe karolong ea II.dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-4Figure 1. proFIRE® chromatogram of a ligand strand conjugate purification. Used buffers: Buffer A [1]; Buffer B [5].
    Column: proFIRE® column. Flow: 1 mL/min. Used program: DNA length 24, Type 1.

Buffer Exchange le Concentration le Centrifugal Filter Units

  1. Take one centrifugal filter unit, add the appropriate volume (500 µL) of buffer in the filter device, and cap it.
  2. Beha sesebelisoa sa filthara se kentsoeng ka har'a rotor ea centrifuge, u lokise leqhama la cap ho ea bohareng ba rotor; kgahlano le sesebedisoa se tshoanang le sona.
  3. Spina sesebelisoa ho 13,000 xg (kapa 14,000 xg) ka nako e fanoeng.
  4. Remove the flow through and repeat steps 1-3 with 350 µL volume.
  5. Tlosa sesebelisoa se bokelitsoeng ho centrifuge 'me u arole sesebelisoa sa filthara ho tloha tube ea microcentrifuge.
  6. Ho khutlisa conjugate, beha sesebelisoa sa filthara fatše ka har'a tube e hloekileng ea centrifugal, u lokise cap e bulehileng ho ea bohareng ba rotor; kgahlano le sesebedisoa se tshoanang le sona. Spin bakeng sa metsotso ea 2 ho 1,000 xg ho fetisetsa sample ho tloha sesebelisoa ho ea ho tube.dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-5

Letlapa la Tšebelisano

Li-additives tsa buffer
Khokahano ea li-ligands ka lisebelisoa tsohle tse fumanehang tsa ho kopanya e ka etsoa ka li-additives tse ngata tse fapaneng. Lenane le latelang le bontša bohle ba lekiloeng, empa ka kopo hlokomela hore ba bang ba sa thathamisoang mona le bona ba ka sebelisoa ka katleho.

dynamic-BIOSENSORS-Y-Structure-Amine-Coupling-Kit-1-fig-6

* li-agent tse fokotsang tse thehiloeng ho thiol
** e na le li-amine tsa mantlha
*** tlhokomeliso, e ka lematsa ligand

pH/pI

Theko ea pH bakeng sa buffer ea conjugation e ka tloha ho pH 5.0 ho ea ho pH 8.0, ho itšetlehile ka litšobotsi tsa ligand. Ha o kopanya liprotheine ka pI ea <6, ka kopo hlokomela hore ho sebelisa buffer e nang le pH e tlase ho ka hlahisa chai e ntle ea conjugate.

Buffer pH Odara No Sebopeho
Phosphate-Citrate Buffer pH 5 - 50 mM buffer letsoai, 150 mm NaCl
Buffer M pH 6.5 BU-M-150-1 50 limilimithara MES, 150 limilimithara NaCl
Buffer A pH 7.2 BU-P-150-10 50 mM Na2HPO4/NaH2PO4, 150 limilimithara NaCl
Buffer C pH 8.0 BU-C-150-1 50 mM Na2HPO4/NaH2PO4, 150 limilimithara NaCl

Letsoai la letsoai
Bakeng sa likhokahano tse tloaelehileng, letsoai la 50 mM buffer le 150 mM NaCl (letsoai le mononovalent) li sebelisoa.
Ha u etsa conjugation ea li-ligands tse matla, etsa bonnete ba hore mahloriso a NaCl a phahame ka ho lekaneng (ho fihlela ho 400 mM NaCl e buelloa). Ho seng joalo, ho ka hlaha pula ea DNA.
The shielding effect of monovalent sodium cations leads to DNA stabilisation through neutralisation of the negative charge on the sugar phosphate backbone.

Linomoro tsa Odara tse Molemo

Lethathamo la 3. Linomoro tsa Taelo

Lebitso la Sehlahiswa Chelete Odara No
Y-sebopeho sa Amine coupling Kit 2 - Botala 3 conjugations HK-NYS-NHS-2
Sefate sa Centrifugal (10 kDa MWCO) Li-5 likhomphutha. CF-010-5
10x Buffer A [1] 50 mL (e fana ka 500 ml) BU-P-150-10
5x Buffer B [5] 50 mL (e fana ka 250 ml) BU-P-1000-5
1x Buffer C [2] 12 mL BU-C-150-1

Ikopanye

Dynamic Biosensors GmbH
Perchtinger Str. 8/10 81379 Munich Jeremane

Bruker Scientific LLC
40 Manning Road, Manning Park Billerica, MA 01821 USA

Lintlha tsa Odara order.biosensors@bruker.com
Tšehetso ea tekheniki support.dbs@bruker.com

www.dynamic-biosensors.com
Lisebelisoa le li-chips li entsoe le ho etsoa Jeremane. ©2025 Dynamic Biosensors GmbH
Bakeng sa Tšebeliso ea Lipatlisiso Feela. Ha e sebelisoe lits'ebetsong tsa tlhahlobo ea bongaka.

  1. Buffer A: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.2
  2. Buffer C: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 8.0
  3. Buffer PE40: 10 mM Na2HPO4/NaH2PO4, 40 mM NaCl, pH 7.4, 0.05 % Tween, 50 µM EDTA, 50 µM EGTA
  4. Bakeng sa ho kopanngoa ha liprotheine tse nang le boima ba molek'hule ho feta 20 kDa: Li-filter tsa Centrifugal tse nang le MWCO ea 10 kDa li ka laeloa bakeng sa ts'ebetso e potlakileng ea mahloriso (Taelo ea No: CF-010-5).
  5. Buffer B: 50 mM Na2HPO4/NaH2PO4, 1 M NaCl, pH 7.2

www.dynamic-biosensors.com

Litokomane / Lisebelisoa

BIOSENSORS Y-Structure Amine Coupling Kit 1 [pdf] Bukana ea Mosebelisi
HK-NYS-NHS-1, Y-Structure Amine Coupling Kit 1, Amine Coupling Kit 1, Coupling Kit 1, Kit 1

Litšupiso

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