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dynamic BIOSENSORS LABELING KIT RED DYE 2 Adapter Stand
Likarolo tsa Bohlokoa
- Ho ngolisoa ha biomolecules > 7 kDa ka dae e nyane e khubelu ea fluorescent ka karabelo e potlakileng le e bonolo.
- Khemistri e tlwaelehileng (NHS ester) e lebisitseng diminerale tsa mantlha (mohlala, NH2-terminus, lysines)
- E kenyelletsa li-reagents bakeng sa likarabelo tse hlano tsa motho ka mong (25 - 200 μg protheine ka 'ngoe)
- Ho ngolisoa ha multiple samples e ka etsoa ka nako e le 'ngoe.
- Tekanyo ea ho ngola (DOL) ka molek'hule e ka fetoloa.
- E lumellana le kanale e khubelu ea lisebelisoa tsohle tsa heliX®.
- Kakaretso ea nako e hlokahalang e ka tlase ho metsotso e 45.
Fetileview Mehato e 3 ea ho Ngola Mosebetsi
- Phapanyetsano ea buffer
Protheine e khahlang e fetisetsoa ho buffer ea lileibole ho sebelisoa likholomo tse fanoeng tsa buffer exchange spin. - Keketso ea dae le ho qhoqhoa
Dae ea fluorescent e kenngoa ho protheine ea thahasello ka tekanyo e lakatsehang ea molar ebe e kenngoa metsotso e 10. - Tlhoekiso
Limolek'hule tsa dae tsa mahala lia tlosoa 'me buffer e fetisetsoa sebakeng se lakatsehang sa polokelo ho sebelisoa litšiea tsa ho tlosa dae tse fanoeng.
Tlhaloso ea Sehlahisoa
Nomoro ea Taelo: CY-LK-R2-1
Lethathamo la 1. Litaba le Boitsebiso ba Polokelo
| Lintho tse bonahalang | Cap | Chelete | Polokelo | Tlhaloso |
| Khubedu dae (NHS ester, e nchafalitsoe ho 2 mM stock ho anhydrous DMSO) | Boputsoa | 5 x 10µL | -20 °C ho ea ho -80°C | sireletsa metsi, moea le khanya, qoba ho qhoqhoa khafetsa ha leqhoa |
| Tšireletso ea ho tšoaea pH 8.3 (15.7 mM carbonate-bicarbonate, 126 mM NaCl,
2.5 mM KCl, 9.2 mM phosphate) |
Tse pepeneneng | 5 x 1 mL | -20 °C | |
| Sebaka sa polokelo DPBS (140 mM NaCl, 2.7 mM KCl, 10 mM phosphate) | Tse pepeneneng | 5 x 1 mL | -20 °C | |
| Kholomo ea phapanyetsano ea buffer (“1”) | Khubedu | 5 x | 2-8 °C | |
| Kholomo ea ho tlosa dae ("2") | Khubedu | 5 x | 2-8 °C | |
| Li-tubes tsa pokello ea 2.0 ml bakeng sa litšiea tsa spin | 10 x | RT | ||
| 1.5 mL li-tubes tsa pokello ea protheine e tlase | 10 x | RT |
Kit e na le li-reagents bakeng sa li-labeling reactions tse 5.
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.
Lihlahisoa li romelloa ka mocheso oa mocheso (RT) 'me li lokela ho buloa ha li fihla le ho bolokoa ho latela likhothaletso tse ho Lethathamo la 1.
Resin slurry ea buffer exchange spin column e na le 0.02 % sodium azide le resin slurry ea dae ea ho tlosa dae e na le 0.05 % sodium azide.
Lisebelisuoa Tse Eketsehileng Tse Hlokehang
Lethathamo la 2. Lisebelisoa tse Eketsehileng
| Lintho tse bonahalang | Tlhaloso |
| Benchtop microcentrifuge | Litlhophiso tsa lebelo tse hlokahalang 1,500 xg le 1,000 g |
| UV-Vis spectroscopy (mohlala, NanoDrop™) | Bakeng sa mahloriso le boikemisetso ba DOL ba conjugate |
Litharollo tse ling tsohle tse hlokahalang, li-buffers le li-tubes li kenyellelitsoe ka har'a kit.
Lintlha Tse Bohlokoa
- O SE KE oa sebelisa li-buffers tse nang le li-amine tsa mantlha (ke hore, Tris, Glycine) nakong ea ho ngola.
- Khatello e tlase ea sodium azide (<3 mM) e tsamaisana le karabelo ea ho ngola.
- Qoba ho pepeseha ha nako e telele dae e sebetsang metsing a metsi, moea le khanya. Ha u fihla, hatsetsa li-aliquots tsa dae tse fanoeng 'me u li boloke ho ≤ -20°C ho fihlela li sebelisoa. Qoba makhetlo a mangata a ho qhibiliha a leqhoa.
Haeba ho hlokahala, hlapolla dae ka kotloloho pele o e sebelisa ho beha li-lailing buffer 'me u se ke ua boloka dae e setseng e hlapolotsoeng. - Pele o qala, ka bokhuts'oane centrifuge lik'hemik'hale tsohle ho etsa bonnete ba hore thepa eohle e ka tlase ho li-tubes.
3-Mehato ea XNUMX ea Fluorescent labeling ea protheine analyte
Ka kopo bala protocol eohle pele u qala le ho etsa mehato eohle ntle le tšitiso. Protocol ena e ka sebelisoa ho tšoaea 25 ho 200 µg ea protheine (concentration lipakeng tsa 0.25 ho 7 mg/ml) ka karabelo ea ho ngola. SampLi-volumes li lokela ho ba lipakeng tsa 30 ho isa ho 120 µl, ntle le palo ea protheine e sebelisitsoeng.
KELETSO Protocol ena e ka etsoa habonolo bakeng sa karabelo ea lileibole tse ngata ka nako e le 'ngoe.
Phapanyetsano ea buffer
- Beakanya kholomo e le 'ngoe ea Buffer exchange spin (“1”) ho latela karabelo ea lileibole ka ho Labela buffer:
a. Tlosa tiiso e ka tlase ea khola 'me u lokolle cap (u se ke ua e tlosa).
b. Beha kholomo ka har'a tube ea pokello ea 2.0 mL.
c. Centrifuge ho 1,500 xg bakeng sa 1 min mocheso oa kamore ho tlosa tharollo ea polokelo.
d. Kenya 400 µL ea Leibole buffer (pH 8.3) kholomong.
e. Centrifuge ho 1,500 xg bakeng sa 1 min ka mocheso oa kamore 'me u lahle phallo-ho feta.
f. Pheta mehato d & e. The Buffer exchange spin column e lokela ho ba boemong bo omileng. - Sample loading:
a. Fetisetsa kholomo ho tube e ncha ea ho bokella liprotheine tse 1.5 mL.
b. Tlosa cap 'me u sebelise ka hloko protheine sample holimo ho bethe ea resin. U se ke ua sitisa bethe ea resin.
c. Centrifuge ho 1,500 xg bakeng sa 2 min ho bokella sample (phallo-ka-ka-). Lahla mokokotlo oa mokokotlo ka mor'a tšebeliso.
Leibole la fluorescent
- Thaw dae aliquot (2 mM stock concentration in anhydrous DMSO) ka kotloloho pele o sebelisoa pele.
- Kenya 1 - 10 µl ea tharollo ea dae ea 2 mM ho liprotheineample ka tekanyo e lakatsehang ea molar 'me u kopanye hang-hang ka ho kenya liphaephe ka hloko holimo le tlase. Sebelisa tafole e latelang e le tataiso bakeng sa ho kenya dae kapa bona example lipalo ho Sehlomathiso bakeng sa lintlha tse ling.
Fab IgG Ho kenya protheine 100 µg ≡ 2 nmol 100 µg ≡ 0.667 nmol Ka makhetlo a 4 molar e feteletseng ea dae 8 nmol 2.7 nmol Setoko sa dae se kentsoeng [2 mM] 4µl 1.33µl - Eketsa motsoako oa karabelo bakeng sa metsotso e 10 mocheso oa kamore. Sireletsa kganya.
Phapanyetsano ea buffer le ho tlosoa ha dae e sa tlamisoang - Ho sa le joalo, lekanya kholomo e le 'ngoe ea ho tlosa Dye (“2”) ho latela karabelo ea lileibole ka Storage buffer (mohlala, PBS):
a. Tlosa tiiso e ka tlase ea khola 'me u lokolle cap (u se ke ua e tlosa).
b. Beha kholomo ka har'a tube ea pokello ea 2.0 mL.
c. Centrifuge ho 1,000 xg bakeng sa 2 min mocheso oa kamore ho tlosa tharollo ea polokelo.
d. Kenya 400 µL ea Storage buffer (DPBS) kholomong.
e. Centrifuge ho 1,000 xg bakeng sa 2 min ka mocheso oa kamore 'me u lahle phallo-ho feta. - Sample loading:
a. Fetisetsa kholomo ea ho tlosa Dye ho tube e ncha ea 1.5 mL ea pokello e tlamang e tlase.
b. Tlosa sekoahelo 'me u sebelise ka hloko protheine e ngotsoeng sample holimo ho bethe ea resin. U se ke ua sitisa bethe ea resin.
c. Centrifuge ho 1,000 xg bakeng sa 2 min ho bokella sample (phallo-through). Dae e sa sebetsanoang e bolokoa kholomong mme e silafatsa boputsoa ba resin. Lahla mohala oa ho tlosa Dye ka mor'a tšebeliso.
Aliquots le Storage - Etsa qeto ea karo-karolelano ea protheine le dae-to-protein (= degree of labeling, DOL), joalo ka ha ho hlalositsoe karolong e latelang.
- Aliquot mme e boloke pakeng tsa 8 °C le -80 °C. Hangata liprotheine tse ngotsoeng li lokela ho bolokoa tlas'a maemo a tšoanang le a liprotheine tse sa ngolisoang.
BOHLOKOA Sireletsa liprotheine tse ngotsoeng khanya.
Sehlomathiso
Boikemisetso ba karo-karolelano ea dae-to-protein (tekanyo ea ho ngola, DOL)
Kakaretso ea tekanyo ea ho ngola e ka baloa ho tloha ho molar concentrations tsa protheine le dae. Lintlha tseo li ka khethoa ho sebelisoa spectroscope ea UV-Vis (mohlala, NanoDrop™).
- Lekanya ho monya ha protheine e ngotsoeng sample ho 643 nm (= A,) ho NanoDrop ka mokhoa oa Protein & Labels (“. Etsa qeto ea hore na dae e na le bokae (Cdye) ka ho kenya boleng ba hau bo lekantsoeng foromong ena:
- Lekanya ho monya ha protheine e ngotsoeng sample ho 280 nm (= Ap) ho NanoDrop '”. Etsa qeto ea bongata ba protheine (= Cp) ka ho kenya litekanyetso tsa hau tse lekantsoeng ho tloha lintlheng tsa 1. le 2. ho foromo ena:
&280 ke coefficient ea ho timela ea protheine ho 280 nm. Bala ka tatellano ea protheine ea hau ka bonngoe kapa ka palo e tloaelehileng:
- &280 ea molek'hule e tloaelehileng ea IgG (~ 150 kDa): 210,000 M-'cm")
- 8280 ea molek'hule e tloaelehileng ea Fab (~ 50 kDa): 74,000 M' cm 1)
- &280 ea Nanobody e tloaelehileng (~15 kDa): 27,000 M'cm”)
- Tekanyo ea ho ngola (DOL) e baloa ka foromo:
Example Palo ea palo ea dae
- Example lipalo bakeng sa li-antibodies tsa IgG
1. Ho ngolla 100 μg ea antibody ea IgG (MW = 150 kDa), e lumellanang le 667 pmol. Optimum analyte volume bakeng sa karabelo ena ke 50 μl, e tsamaellanang le khatello ea pele ea analyte ea 2.0 mg/ml. Leha ho le joalo, sampLi-volume tse pakeng tsa 30 μl le 120 μl li ka sebelisoa.
2. Kamora ho fapanyetsana ha buffer ho Leibole buffer, dae ea fluorescent e eketsoa ka kotloloho ho lesole la 'mele. E le hore u finyelle DOL ea 1 - 2, ho lokela ho eketsoa ka makhetlo a 4 ho feta molar ea dae. Sena se lumellana le 2.67 nmol ea dae, e lumellanang le 1.33 μl ea 2 mm ea tharollo ea stock. Ntle le moo, bakeng sa pipetting e nepahetseng haholoanyane ea molumo o monyenyane, dae e ka hlapolloa 1:10 ka Leibole buffer ka kotloloho pele e sebelisoa ebe 13.3 μl ea tharollo ea 0.2 mM e sebelisoa bakeng sa karabelo ea ho ngola.
3. Mohlahlobi oa ho qetela, o hloekisitsoeng o ngotsoeng o lokela ho bonts'a ka mokhoa o nepahetseng maikutlo> 2 μM le DOL pakeng tsa 1 le 2. - Example lipalo bakeng sa likhechana tsa Fab
1. Ho ngolisoa ha 100 μg ea Fab fragment (MW = 50 kDa), e lumellanang le 2 nmol. Optimum analyte volume bakeng sa karabelo ena ke 50 μl, e tsamaellanang le khatello ea pele ea analyte ea 2.0 mg/ml. Leha ho le joalo, sampLi-volume tse pakeng tsa 30 μl le 120 μl li ka sebelisoa.
2. Kamora ho fapanyetsana ha buffer ho Leibole buffer, dae ea fluorescent e eketsoa ka kotloloho ho lesole la 'mele. E le hore u finyelle DOL ea 1 - 2, ho lokela ho eketsoa ka makhetlo a 4 ho feta molar ea dae. Sena se lumellana le 8 nmol ea dae, e lumellanang le 4 μl ea 2 mm ea tharollo ea stock. Haeba ho hlokahala ho etsa hore palo e nyane ea liphaephe e nepahale, dae e ka hlapolloa ka 1:10 ka "Labeling buffer" pele e sebelisoa.
3. Mohlahlobi oa ho qetela, o hloekisitsoeng o ngotsoeng o lokela ho bonts'a ka mokhoa o nepahetseng maikutlo> 6 μM le DOL pakeng tsa 1 le 2.
Ho batle phoso
Lintlha tse akaretsang: Molek’hule o le mong oa protheine o ka ngoloa ka molek’hule e le ’ngoe kapa tse ngata tsa dae. Karo-karolelano e phahameng ea dae ho protheine e ka eketsa matla a lets'oao empa hape e eketsa monyetla oa ho bokellana ha protheine le phetoho ea sebaka se tlamang. Mekhoa ea ho ngola ka kakaretso e lokela ho ikemisetsa ho hlahisa molek'hule e le 'ngoe ho isa ho tse peli tsa dae ka molek'hule ea protheine. Litlhahlobo tse nang le kamano e tlase e tlamang li ka tlameha ho sebelisoa maemong a holimo nakong ea litekanyo tsa switchSENSE® le Real-time Interaction Cytometry (RT-IC), tse lebisang ho lets'oao le phahameng. Tabeng ena, mekhoa ea ho ngola e lokela ho ikemisetsa ho fokotsa DOL ea 0.5 - 1.
Tafole ea 3. Tharollo ea mathata
| Ke kopane le bothata | Tlhaloso e ka bang teng | Ntlafatso |
| Kotulo e tlaase (concentration) ea analyte e ngotsoeng ka mor'a ho hloekisa | Motsoako o tlase oa tharollo ea analyte e se e ntse e le teng pele ho karabelo ea ho ngola | Ho tsepamisa mohopolo oa protheine analyte pele ho laibole ho sebelisa li-filters tsa centrifugal (sheba karolo ea Linomoro tse U thusang). |
| DOL e tlase ka mor'a ho hloekisoa | Khatello e tlase ea tharollo ea analyte pele karabelo ea ho ngola e ka fokotsa ts'ebetso ea karabelo ea ho ngola le ho khothaletsa hydrolysis ea dae ea NHS ester ka tharollo ea metsi. | Ho tsepamisa mohopolo oa protheine analyte pele ho laibole ho sebelisa li-filters tsa centrifugal (sheba karolo ea Linomoro tse U thusang). |
| Khatiso ea dae e tlase haholo | Eketsa molar e feteletseng ea dae ho fihlela makhetlo a 10. | |
| Nako ea ho arabela e khuts'oane haholo | Eketsa nako ea ho arabela ho fihla ho 1h ho RT. | |
| Sample na le litšila, litšila kapa lijari (mohlala, BSA) | Ntlafatsa le ho netefatsa bohloeki ba analyte u sebelisa mokhoa o hlophisitsoeng oa khetho. Haeba bohloeki ba analyte bo ke ke ba ntlafatsoa, eketsa molar e feteletseng ea dae ho fihlela makhetlo a 10. | |
| Analyte e ngolisitsoeng e ntse e tsitsa (ho senoleha kapa ho bonoa sebopeho se kopaneng) | DOL e phahame haholo | Fokotsa molar e feteletseng ea dae ho 1-fold, kapa fokotsa nako ea ho arabela ho metsotso e 5, kapa fokotsa mocheso oa ho arabela ho 4 ° C. |
| Analyte e na le maikutlo a pH | Etsa karabelo ea ho ngola ho Storage buffer (PBS) kapa buffer e loketseng ea khetho (pH ≥ 7.2). Sena se ka fokotsa katleho ea karabelo ea ho ngola le sephetho sa analyte DOL. The molar feteletseng ea dae e ka eketsoa ho fihlela makhetlo a 10 kapa nako ea karabelo e ka eketsoa ho fihla ho 1 h ho hanyetsa phello ena. | |
| Analyte e khona ho mamella mocheso | Etsa karabelo ea ho ngola ka 4 °C. Sena se ka fokotsa katleho ea karabelo ea ho ngola le sephetho sa analyte DOL. The molar feteletseng ea dae e ka eketsoa ho 10-fold kapa nako ea karabelo e ka eketsoa ho fihla ho 16 h ho hanyetsa phello ena. |
Linomoro tsa Odara tse Molemo
Lethathamo la 4. Linomoro tsa Taelo
| Lebitso la sehlahisoa | Nomoro ea odara |
| 10 x DPBS | BU-DP-10-1 |
| 10 x RT-IC Running Buffer 1 | BU-RB-10-1 |
| Setsi sa filtara ea Centrifugal (3 kDa MWCO), li-pcs tse 5. | CF-003-5 |
| Setsi sa filtara ea Centrifugal (10 kDa MWCO), li-pcs tse 5. | CF-010-5 |
Ikopanye
Dynamic Biosensors GmbH
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81379 Munich
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Woburn, MA 01801
usa
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dynamic BIOSENSORS LABELING KIT RED DYE 2 Adapter Stand [pdf] Bukana ea Mosebelisi CY-LK-R2-1, v1.2, LABELING KIT RED DYE 2 Adapter Stand, LABELING KIT RED DYE 2, Adapter Stand, Stand, Adapter |