中譜橙化學(xué)發(fā)光磁珠

(尺寸1.0um、2.8um和4.5um)

中譜橙 Homo-COOH 羧基磁珠

　　一、概述

　　中譜橙 Homo-COOH磁珠提供永久結(jié)合和固定各種含胺基配體。 珠子表面上的高密度羧酸基團(tuán)(COOH)可用于配體如蛋白質(zhì)、肽和核酸的共價(jià)偶聯(lián)。 用EDC活化珠表面上的羧基，使配體通過伯胺基與磁珠偶聯(lián)，形成穩(wěn)定的酰胺鍵。 中譜橙 Homo-COOH是均勻的PS/GMA共聚超順磁珠，適用于各種磁性分離應(yīng)用，包括親和力富集、蛋白質(zhì)純化、免疫沉淀和細(xì)胞分選。

　　二、產(chǎn)品特性

　　1. 中譜橙 HomoS-COOH短鏈羧基磁珠

　　

 

　　基團(tuán)密度> 400 umoles carboxylic acid/g beads

　　2. 中譜橙 HomoL-COOH長(zhǎng)鏈羧基磁珠

　　

 

　　基團(tuán)密度> 200 μmoles carboxylic acid/g beads

　　形態(tài)：超順磁球形

　　直徑：1μm

　　儲(chǔ)存：10 mg/mL ，去離子水中 2~8 °C保存，勿凍結(jié)。

　　常溫運(yùn)輸，正確使用保存期一年。

　　三、緩沖液

　　Coupling Buffer(偶聯(lián)緩沖液):

　　50 mM MES [2-(N-morpholino)-ethanesulfonic acid], pH 6.0, 0.01% Triton X-100;

　　Coupling Agent(偶聯(lián)試劑):

　　EDC [1-ethyl-3-(3-dimethyaminopropyl)-carbodiimide];

　　Sulfo-NHS [N-hydroxysulfosuccinimide] (Optional)

　　Quench Buffer(淬滅緩沖液):

　　TBS (25 mM Tris-Cl, 130 mM NaCl, 2.7 mM KCl), pH 8, 0.01% Triton X-100;

　　Storage Buffer(儲(chǔ)存緩沖液):

　　TBS or PBS containing 0.01% Triton X-100 or 0.01% Tween 20. Add preservative if needed.

　　四、中譜橙 Homo-COOH的使用

　　以下方案為配體與100μL的中譜橙 Homo -COOH羧基磁珠的偶聯(lián)方案。該方案可按需放大或縮小。強(qiáng)烈建議對(duì)珠子活化(EDC、EDC / NHS和pH)和偶聯(lián)條件(配體濃度、偶聯(lián)緩沖液、pH、反應(yīng)體積和溫育時(shí)間)進(jìn)行優(yōu)化。

　　注意：

　　■ 通過渦旋確保磁珠均勻懸浮。

　　■ 為獲得最佳性能，在無(wú)胺偶聯(lián)緩沖液中偶聯(lián)，蛋白應(yīng)為0.5?4mg / mL，寡核苷酸為20?100μM，不含其它蛋白。較高濃度的蛋白可靈活地優(yōu)化反應(yīng)體積。含tris、甘氨酸、醋酸鹽或檸檬酸鹽的緩沖液不能使用。

　　■ 含有0.01%的Triton X-100的50 mM MES緩沖液(pH 6.0)可用作活化和偶聯(lián)緩沖液。當(dāng)使用化學(xué)合成的寡核苷酸時(shí)，寡核苷酸末端應(yīng)有5'-胺基以偶聯(lián)磁珠。

　　■ 珠子含有0.5%SDS以穩(wěn)定懸浮液。盡管非必須，但在所有緩沖液中包含0.01?0.05%Triton X-100或0.01?0.05%吐溫20將防止珠粒凝結(jié)并改善分散特性。

　　偶聯(lián)方案A：使用EDC的兩步偶聯(lián)

　　該方案被推薦用于將蛋白偶聯(lián)到羧基磁珠上。首先使用水溶性碳二亞胺(EDC)活化珠上的羧基以形成胺反應(yīng)性中間體。除去EDC后，加入蛋白質(zhì)配體并通過蛋白質(zhì)上的伯胺與磁珠的活化羧基偶聯(lián)。

　　1. 確保蛋白或配體在無(wú)胺偶聯(lián)緩沖液中。100μL偶聯(lián)緩沖液中需50?400μg蛋白用于耦合。放在冰上。

　　2. 渦旋振蕩重懸中譜橙 Homo-COOH羧基磁珠。吸取100μL磁珠到1.5mL EP管中，磁分離并吸棄上清。

　　3. 加入200μL偶聯(lián)緩沖液，渦旋20秒洗滌磁珠，磁分離并吸棄上清。

　　4. 按步驟3再次清洗羧基磁珠兩次。

　　5. 使用前用偶聯(lián)緩沖液中配制EDC(50 mg / mL)。

　　6. 向磁珠中加入80μL偶聯(lián)緩沖液和20μL新配的EDC溶液，混勻。室溫孵育15分鐘，磁分離并吸棄上清。

　　7. 用200μL偶聯(lián)緩沖液清洗磁珠，磁分離并吸棄上清。

　　注意：該步驟應(yīng)該快速進(jìn)行，因?yàn)榇胖樯系陌贩磻?yīng)性中間體不穩(wěn)定。

　　8. 向羧基磁珠加入100μL偶聯(lián)緩沖液和50?400μg蛋白質(zhì)或配體，渦旋混勻。

　　9. 室溫下孵育30分鐘。根據(jù)配體和濃度的不同，最佳孵育時(shí)間可能為0.5?4小時(shí)。

　　10. 將試管放入磁力架中，磁分離并吸棄上清。該上清液含有未結(jié)合的配體，如果優(yōu)化方案可保存用于分析。

　　11. 向羧基磁珠加入500μL淬滅緩沖液。渦旋20秒，磁分離并吸棄上清。

　　12. 向羧基磁珠加入500μL淬滅緩沖液。室溫孵育30?60分鐘。磁分離并吸棄上清。

　　13. 向羧基磁珠加入500μL淬滅緩沖液。劇烈漩渦20秒，磁分離并吸棄上清。按該步驟再洗兩次磁珠。

　　14. 從磁力架上取下EP管。添加100μL存儲(chǔ)緩沖液。渦旋混合并在2?8°C儲(chǔ)存偶聯(lián)好的磁珠。

　　偶聯(lián)方案B：用sulfo-NHS替代兩步偶聯(lián)

　　該方案與上述方案類似，但使用NHS?？赏ㄟ^加入穩(wěn)定胺反應(yīng)性中間體的sulfo-NHS來(lái)增加EDC介導(dǎo)的反應(yīng)的效率。

　　1. 確保蛋白或配體在無(wú)胺偶聯(lián)緩沖液中。100μL偶聯(lián)緩沖液中需50?400μg蛋白用于耦合。放在冰上。

　　2. 渦旋振蕩重懸中譜橙 Homo-COOH羧基磁珠。吸取100μL磁珠到1.5mL的EP管中，磁分離吸棄上清。

　　3. 加入200μL偶聯(lián)緩沖液。劇烈漩渦20秒，磁分離并吸棄上清。

　　4. 按步驟3再清洗羧基磁珠兩次。

　　5. 使用前用偶聯(lián)緩沖液中配制EDC(50 mg / mL)。在使用前用偶聯(lián)緩沖液配制Sulfo-NHS(50mg / mL)。

　　6. 向磁珠中加入60μL偶聯(lián)緩沖液、20μL新配的EDC溶液和20μL新配的Sulfo-NHS溶液。渦旋混勻。

　　7. 室溫下混勻孵育15分鐘。磁分離并吸棄上清。

　　8. 用200μL偶聯(lián)緩沖液清洗磁珠。渦旋混勻，磁分離并吸棄上清。

　　9. 加入100μL偶聯(lián)緩沖液和50?400μg蛋白或配體。渦旋混勻。在室溫下連續(xù)混合孵育0.5?4小時(shí)。

　　10. 將試管放入磁力架中，磁分離并吸棄上清。該上清液含未結(jié)合的配體，如果優(yōu)化方案可保存用于分析。

　　11. 向羧基磁珠加入250μL淬滅緩沖液。渦旋20秒，磁分離并吸棄上清。

　　12. 向羧基磁珠加入500μL淬滅緩沖液。室溫孵育30?60分鐘。磁分離并吸棄上清。

　　13. 向羧基磁珠加入250μL淬滅緩沖液。劇烈漩渦20秒，磁分離并吸棄上清。

　　14. 從磁力架上取下EP管。添加100μL存儲(chǔ)緩沖液。渦旋混合并在2?8°C儲(chǔ)存偶聯(lián)好的磁珠。

　　偶聯(lián)方案C：一步偶聯(lián)

　　這是一個(gè)快速結(jié)合方案，在一個(gè)反應(yīng)??中同時(shí)加入羧基磁珠、EDC和配體。該方案最適合偶聯(lián)寡核苷酸和小分子，當(dāng)配體上的羧酸基團(tuán)的激活不會(huì)影響后續(xù)實(shí)驗(yàn)時(shí)。

　　1. 確保蛋白或配體在無(wú)胺偶聯(lián)緩沖液中。100μL偶聯(lián)緩沖液中需50?400μg蛋白或1?5nmol寡核苷酸進(jìn)行偶聯(lián)。放在冰上。

　　2. 渦旋振蕩重懸中譜橙 Homo-COOH羧基磁珠。吸取100μL磁珠到1.5mL的EP管中，磁分離吸棄上清。

　　3. 加入200μL偶聯(lián)緩沖液。劇烈漩渦20秒，磁分離并吸棄上清。

　　4. 按步驟3再清洗羧基磁珠兩次。

　　5. 從磁力架上取下EP管。在80μL偶聯(lián)緩沖液中加入50?400μg配體。

　　6. 在室溫下孵育30分鐘。

　　7. 使用前用偶聯(lián)緩沖液配制EDC(50 mg / mL)。

　　8. 將20μL新配的EDC溶液加入磁珠中。渦旋混勻，室溫下連續(xù)混合孵育0.5?4小時(shí)。

　　9. 將試管放入磁力架中，磁分離并吸棄上清。該上清含未結(jié)合的配體，如果優(yōu)化方案可以保存用于分析。

　　10. 向羧基磁珠加入500μL淬滅緩沖液。渦旋20秒，磁分離并吸棄上清。

　　11. 向羧基磁珠加入500μL淬滅緩沖液。室溫孵育30?60分鐘。磁分離并吸棄上清。

　　12. 向羧基磁珠加入500μL淬滅緩沖液。劇烈漩渦20秒，磁分離并吸棄上清。按該步驟再洗兩次磁珠。

　　13. 從磁力架上取下EP管。添加100μL存儲(chǔ)緩沖液。渦旋混合并在2?8°C儲(chǔ)存偶聯(lián)好的磁珠。

中譜橙? Homo-Streptavidin Beads

(鏈酶親和素磁珠)

　　中譜橙? Homo-Streptavidin (SA) Beads are ideal for nucleic acid diagnostics, specifically with samples with a high chaotropic salt concentration, immunoassays involving small biotinylated antigens and applications that are not compatible with BSA (these beads are not blocked with BSA). Homo-Streptavidin Beads offer increased binding capacity and slower sedimentation rate.

　　Add SA Beads to a sample containing biotinylated molecules such as peptides, oligonucleotides etc. During a short incubation, the biotinylated molecule will bind to the beads. Separate the molecule-bead complex with a magnet. Capture, washing and detection can be optimized for manual or automated use. With indirect capture, mix the biotinylated molecule with the sample to capture the molecule target complex before adding beads. Indirect target capture is an advantage when molecule-target kinetics are slow, affinity is weak, molecule concentration is low or molecule target binding requires optimal molecule orientation and true liquid-phase kinetics. Due to their high surface area per weight, uniformity, excellent batch reproducibility and ease of adaptation to automated processes, 中譜橙 beads have become the solid phase of choice for developing immunoassays (/IVD).

　　Product information

　　Diameter: 1 μm (Core-shell GMA particle)

　　Concentration: 10 mg/ml

　　Ligand: Streptavidin

　　Type Magnetization: Superparamagnetism

　　Store at 2-8°C(up to 6 months)in Storage Buffer (25 mM tris-HCl, 0.15 M NaCl, 0.05% tween20, 0.05% NaN3, pH 7.2). Do not freeze the reagent.

　　Binding capacity

 

Free Biotin	> 2500 pmol
Biotinylated peptides	~ 400 pmol
Biotinylated antibody	up to 20 pmol
ds DNA	~ 20μg
ss oligonucleotides	~ 500 pmol
* Oligonucleotides and DNA fragments

 

　　Recommended buffers and solutions

 

For coupling of Nucleic Acids

For beads treatment before RNA manipulations

For coupling of protein or other molecules

 

　　How to use this product

　　Critical Notes

　　In the protocols we recommend keeping the tube on the magnet for up to 2 mins to ensure that all the beads are collected on the tube wall. For non-viscous samples, separation is often complete in under 1 min, once you can see the beads collected.

　　For diluted samples increase the incubation time or isolate in smaller batches using the same beads in each batch.

　　Use a mixer to tilt/rotate the tubes so 中譜橙 beads do not settle at the tube bottom.

　　Avoid air bubbles during pipetting. Free biotin in the sample will reduce the binding capacity of the beads. A disposable separation column or a spin column will remove unincorporated biotin.

　　Run the PCR with limiting concentrations of biotinylated primer, or remove free biotinylated primer by ultrafiltration, microdialysis or other clean-up protocols. PCR Clean Up products are available from.

　　Both the size of the molecule to be immobilized and the biotinylation procedure will affect the binding capacity. Large as well as small biotinylated molecules can be immobilized. The capacity for biotinylated molecules depends on steric availability and charge interaction between bead and molecule and between molecules. There are two or three biotin binding sites available for each streptavidin molecule on the surface of the bead after immobilization.

　　Optimize the quantity of beads used for each individual application by titration. Use up to two-fold excess of the binding capacity of the biotinylated molecule to saturate streptavidin. Binding efficiency can be determined by comparing molecule concentration before and after coupling.

　　Immobilization Procedure

　　■ Bead Preparation

　　1) Resuspend the beads in the original vial.

　　2) Calculate the amount of beads required based on their binding capacity and transfer the beads to a new tube.

　　3) Wash 中譜橙 beads to remove preservatives.

　　nucleic acid applications: 1x B&W Buffer

　　antibody/protein applications: PBS, pH 7.4

　　■ Washing Procedure

　　4) Place the tube containing the beads on a magnet for 1-2 mins.

　　5) Remove the supernatant by aspiration with a pipette while the tube is on the magnet.

　　6) Remove the tube from the magnet.

　　7) Add washing buffer along the inside of the tube where the beads are collected and Resuspend (same volume of washing buffer as the initial volume of beads taken from the vial or larger).

　　8) Repeat steps 4 to 7 twice, for a total of 3 washes.

　　If using 中譜橙 beads for RNA Manipulation:

　　As 中譜橙 beads Streptavidin are NOT supplied in RNase-free solutions, perform the following steps after washing for RNA applications:

　　9) Wash the beads twice in Solution A for 2 mins. Use the same volume of beads as recommended in step 7.

　　10) Wash the beads once in Solution B. Use the same volume of beads as in step 9.

　　11) Resuspend the beads in Solution B.

　　The beads are now ready to be coated with the biotinylated molecule of your choice.

　　■ General Immobilization Protocol

　　Wash the 中譜橙 beads according to section above before use.

　　1) Add the biotinylated molecule to the washed 中譜橙 beads.

　　2) Incubate for 15-30 min at room temperature with gentle rotation of the tube.

　　3) Place the tube in a magnet for 2-3 mins and discard the supernatant.

　　4) Wash the beads 3-4 times in washing buffer.

　　5) Resuspend to desired concentration in a suitable buffer for your downstream use. Here are some examples of immobilization protocols for specific applications.

　　■ Immobilization of Nucleic Acids

　　1) Resuspend beads in 2x B&W Buffer to a final concentration of 5 μg/μl (twice original volume).

　　2) To immobilize, add an equal volume of the biotinylated DNA/RNA in H2O to dilute the NaCl concentration in the 2x B&W Buffer from 2M to 1 M for optimal binding.

　　3) Incubate for 15 mins at room temperature using gentle rotation. Incubation time depends on the nucleic acid length: short oligonucleotides (< 30 bases) require max. 10 mins. DNA fragments up to 1 kb require 15 mins.

　　4) Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins.

　　5) Wash 2–3 times with a 1x B&W Buffer.

　　6) Resuspend to the desired concentration. Binding is now complete. Resuspend the beads with the immobilized DNA/RNA fragment in a buffer with low salt concentration, suitable for downstream applications.

　　■ Release of Immobilized Biotinylated Molecules

　　The biotin-streptavidin bond is broken by harsh conditions. 5 mins incubation at 65°C or 2 mins at 90°C in 10 mM EDTA pH 8.2 with 95% formamide will typically dissociate >96% of immobilized biotinylated DNA. Alternatively, boil the sample for 5 mins in 0.1% SDS for protein dissociation.

　　Please note that proteins will be denatured by such treatment and 中譜橙 beads Streptavidin cannot be re-used. It has also been reported that the biotin-streptavidin interaction can be broken by a short incubation in nonionic water at a temperature above 70°C.