Lightning-Link® Cy3® 抗體標定試劑盒

Cy3® Conjugation Kit (Fast) - Lightning-Link®

貨號

ab188287

包裝

3 x 10 μg
100 μg
3 x 100 μg
1 mg

廠牌

Abcam

產品簡介

Abcam Cy3® Conjugation Kit (Fast) - Lightning-Link® 提供了一種簡便快速的方法，可將 Cy3® 螢光染料標定至抗體或其他含有游離胺基的蛋白質、胜肽分子上，標定產物可直接應用於 Western Blot、ELISA、免疫組織化學染色 (IHC) 等實驗。套組採用 Lightning-Link® 直接標定技術，僅需將修飾劑 (Modifier) 加入待標定抗體，再以此溶液回溶凍乾的 Cy3® 試劑小瓶，即可啟動標定反應；動手操作時間約 30 秒，20 分鐘左右即可完成標定，全程無需任何純化步驟，抗體回收率可達 100%。

Abcam Lightning-Link® Conjugation Kit

主要特色

  • 操作極為快速，動手操作時間僅約 30 秒，全程約 20 分鐘即可完成。
  • 全程無需純化步驟，抗體回收率可達 100%，標定完成後即可直接應用於 Western Blot、ELISA、IHC 等實驗，無需額外處理。
Fluorescence Resonance Energy Transfer (FRET). Mellal, Katia, et al used Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as part of examining effects of MPE-001 on the CD36/TLR2 interaction. They used the kit to conjugate Cy3® to anti-TLR2 antibody for use in FRET. Peritoneal MPs were stimulated with 300 ng/ml R-FSL1 in the presence of 10-7M MPE-001 or vehicle. (A) MPE-001 disrupted the interaction between CD36 labeled with Cy5 (red) using the Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288) and TLR2 labeled with Cy3® (green) using the Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as assessed by FRET after 5 min stimulation with R-FSL1. (B) Percentage of energy transfer measured using LSM-700 confocal microscope (Zeiss). One-way ANOVA test with Newman-Keuls post-test for multiple comparison was performed. *P<0.05, **P<0.01 and ***P<0.001 vs R-FSL1. Data are shown as mean ±S.E.M.
Fluorescence Resonance Energy Transfer (FRET). Mellal, Katia, et al used Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as part of examining effects of MPE-001 on the CD36/TLR2 interaction. They used the kit to conjugate Cy3® to anti-TLR2 antibody for use in FRET. Peritoneal MPs were stimulated with 300 ng/ml R-FSL1 in the presence of 10-7M MPE-001 or vehicle. (A) MPE-001 disrupted the interaction between CD36 labeled with Cy5 (red) using the Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288) and TLR2 labeled with Cy3® (green) using the Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as assessed by FRET after 5 min stimulation with R-FSL1. (B) Percentage of energy transfer measured using LSM-700 confocal microscope (Zeiss). One-way ANOVA test with Newman-Keuls post-test for multiple comparison was performed. *P<0.05, **P<0.01 and ***P<0.001 vs R-FSL1. Data are shown as mean ±S.E.M. Image from Mellal et al., Sci rep., 9(1):12903. doi: 10.1038/s41598-019-49472-8. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Immunohistochemistry. Hausmann, Annika, et al used Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as part of examining flagella expression in mice orally infected with Salmonella Typhimurium (S. Tm). They used the kit to conjugate Cy3® to anti-FliC antibody for use in immunohistochemistry. Intestinal epithelial cell (IEC)-specific deletion of NAIP1-6 (Naip1-6/IEC; open circles; ntotal S. Tm=776) leads to an increase of flagellated S. Tm at 12 hpi within the cecal mucosa (Naip1-6fl/fl; circles; ntotal S. Tm=238). Quantifications are based on immunofluorescence staining for the flagella subunit FliC. Black bar, median. Statistical analysis, Mann-Whitney-U Test, p-value indicated. Scale bar, 10 μm.
Immunohistochemistry. Hausmann, Annika, et al used Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as part of examining flagella expression in mice orally infected with Salmonella Typhimurium (S. Tm). They used the kit to conjugate Cy3® to anti-FliC antibody for use in immunohistochemistry. Intestinal epithelial cell (IEC)-specific deletion of NAIP1-6 (Naip1-6/IEC; open circles; ntotal S. Tm=776) leads to an increase of flagellated S. Tm at 12 hpi within the cecal mucosa (Naip1-6fl/fl; circles; ntotal S. Tm=238). Quantifications are based on immunofluorescence staining for the flagella subunit FliC. Black bar, median. Statistical analysis, Mann-Whitney-U Test, p-value indicated. Scale bar, 10 μm. Image from Hausmann et al., Mucosal Immunol., 13(3):530-544; doi: 10.1038/s41385-019-0247-0. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Fluorescence Microscopy. Zhang, Fei, Yuan Xie, and Yuhao Bian used Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as part of examining cardiomyogenic differentiation of adipose-derived stem cells. They used the kit to conjugate TGF-β1 protein to Cy3® for use in confocal microscopy. Confocal microscopy was performed to analyze (A, B) TGF-1 binding to rTGF-1 RI on ADSC cell membranes and to visualize (C, D) the colocalization of rTGF-β1 RI (DMPE-PEG-TGF-β1 RI-FITC) and TGF-1 (Cy3-TGF-1) on the surface of ADSCs at 64 oil immersion (scale bar, 100 μm; *p<0.05, **p<0.01, ***p<0.001).
Fluorescence Microscopy. Zhang, Fei, Yuan Xie, and Yuhao Bian used Cy3® Conjugation Kit (Fast) - Lightning-Link® (ab188287) as part of examining cardiomyogenic differentiation of adipose-derived stem cells. They used the kit to conjugate TGF-β1 protein to Cy3® for use in confocal microscopy. Confocal microscopy was performed to analyze (A, B) TGF-1 binding to rTGF-1 RI on ADSC cell membranes and to visualize (C, D) the colocalization of rTGF-β1 RI (DMPE-PEG-TGF-β1 RI-FITC) and TGF-1 (Cy3-TGF-1) on the surface of ADSCs at 64 oil immersion (scale bar, 100 μm; *p<0.05, **p<0.01, ***p<0.001). Image from Zhang et al., Stem Cell Res Ther., 9(1):358; doi: 10.1186/s13287-018-1090-z. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

實驗注意事項

  • 樣本緩衝液 pH 值須介於 6.5–8.5，相容成分包括：Tris（濃度應 ≤50 mM / 0.6%）、BSA（≤0.1%）、甘油 (glycerol，≤50%)、疊氮化鈉 (Sodium Azide，≤0.1%)、磷酸鉀、氯化鈉、蔗糖、檸檬酸鈉、海藻糖等。緩衝液不可含有 Thimerosal (Thiomersal)、Proclin、Glycine、Arginine、Glutathione 等成分，以免干擾標定反應。更多樣本緩衝液細部條件說明，可參照下方表格或產品說明書。
  • 僅適用於已純化的抗體樣本；若樣本中含有其他蛋白質、胜肽或游離胺基分子，可能會競爭消耗標定試劑，影響標定效率。抗體樣本理想濃度為 1 mg/mL，若濃度不足（< 0.5 mg/ml）或過高（> 2 mg/ml），建議先進行濃縮或稀釋處理。
  • 標定完成的產物於 4°C 保存可穩定達 18 個月；若需長期保存，建議加入 50% 甘油作為冷凍保護劑，並存放於 -20°C。實際最適合的保存條件仍視個別抗體的穩定性而定，建議透過實驗確認。
Compatible buffer constituents
50mM / 0.6% Tris10.1% BSA250% glycerol
0.1% sodium azidePBSPotassium phosphate
Sodium chlorideHEPESSucrose
Sodium citrateEDTATrehalose
1 Tris buffered saline is almost always ≤ 50 mM / 0.6%
2 BSA can also interfere with the use of the conjugated antibody in tissue staining.
Incompatible buffer constituents
ThiomerosalProclinGlycine
ArginineGlutathioneDTT

套組內容物

Item3 x 10 µg100 µg3 x 100 µg1 mgStorage temperature
Cy3® mix3 vials1 vial3 vials1 vial-20°C
Modifier reagent1 vial1 vial1 vial1 vial-20°C
Quencher reagent1 vial1 vial1 vial1 vial-20°C

產品說明書

ab188287 Cy3® Conjugation Kit (Fast) - Lightning-Link®
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