Lightning-Link® Alexa Fluor® 488 抗體標定試劑盒

Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link®

貨號

ab236553

包裝

3 x 10 μg
100 μg
3 x 100 μg
1 mg

廠牌

Abcam

產品簡介

Abcam Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® 提供了一種簡便快速的方法，可將 Alexa Fluor® 488 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、流式細胞分析 (flow cytometry)、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 Alexa Fluor® 488 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、Flow cytometry、ELISA、IHC 等實驗，無需額外處理。
Immunocytochemistry. Direct immunofluorescent staining using Recombinant Anti-Ki67 antibody [EPR3610] - BSA and Azide free (ab209897) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553). Lightning-Link® Alexa Fluor® 488 Anti-Ki67 antibody [EPR3610] conjugate was used to stain Ki67-wild-type HAP1 cells (top panel) and Ki67-knock-out HAP1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-Ki67 Alexa Fluor® 488 (shown in green) or ab209897 (Anti-Ki67 antibody, unlabelled control) overnight at +4°C. ab209897 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Immunocytochemistry. Direct immunofluorescent staining using Recombinant Anti-Ki67 antibody [EPR3610] - BSA and Azide free (ab209897) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553). Lightning-Link® Alexa Fluor® 488 Anti-Ki67 antibody [EPR3610] conjugate was used to stain Ki67-wild-type HAP1 cells (top panel) and Ki67-knock-out HAP1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-Ki67 Alexa Fluor® 488 (shown in green) or ab209897 (Anti-Ki67 antibody, unlabelled control) overnight at +4°C. ab209897 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Immunocytochemistry. Direct immunofluorescent staining using Recombinant Anti-Cytokeratin 19 antibody [EP1580Y] - BSA and Azide free (ab195872) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553). Lightning-Link® Alexa Fluor® 488 Anti-Cytokeratin 19 [EP1580Y] conjugate was used to stain wild-type MCF7 cells (top panel) and A431 negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-Cytokeratin 19 Alexa Fluor® 488 (shown in green) or ab195872 (Anti-Cytokeratin 19 antibody, unlabelled control) overnight at +4°C. ab195872 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Immunocytochemistry. Direct immunofluorescent staining using Recombinant Anti-Cytokeratin 19 antibody [EP1580Y] - BSA and Azide free (ab195872) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553). Lightning-Link® Alexa Fluor® 488 Anti-Cytokeratin 19 [EP1580Y] conjugate was used to stain wild-type MCF7 cells (top panel) and A431 negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-Cytokeratin 19 Alexa Fluor® 488 (shown in green) or ab195872 (Anti-Cytokeratin 19 antibody, unlabelled control) overnight at +4°C. ab195872 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Flow Cytometry. Bardhan, Kankana, et al used Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553) as part of examining PD-1pY248+ (pPD-1) expression in human T cells. They used the kit to conjugate Alexa Fluor® 488 to Anti-PD-1pY248 antibody for use in flow cytometry. pPD-1 is predominantly expressed in CD8+ TCM cells. CCR7 and CD45RO markers were used to identify central memory (TCM) and effector memory (TEM) T cells. After gating on CD45RO expression, TCM (CD45RO+CCR7+) and TEM (CD45RO+CCR7−) CD4+ and CD8+ T cells were identified by assessing expression of CCR7. In TCM and TEM populations, expression of PD-1 was determined and, subsequently, expression of pPD-1 (pPD-1-Y248) was assessed in the PD-1+ population within each subset. Results are representative of six separate experiments.
Flow Cytometry. Bardhan, Kankana, et al used Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553) as part of examining PD-1pY248+ (pPD-1) expression in human T cells. They used the kit to conjugate Alexa Fluor® 488 to Anti-PD-1pY248 antibody for use in flow cytometry. pPD-1 is predominantly expressed in CD8+ TCM cells. CCR7 and CD45RO markers were used to identify central memory (TCM) and effector memory (TEM) T cells. After gating on CD45RO expression, TCM (CD45RO+CCR7+) and TEM (CD45RO+CCR7−) CD4+ and CD8+ T cells were identified by assessing expression of CCR7. In TCM and TEM populations, expression of PD-1 was determined and, subsequently, expression of pPD-1 (pPD-1-Y248) was assessed in the PD-1+ population within each subset. Results are representative of six separate experiments. Image from Bardhan, Kankana, et al., Scientific reports, 9(1):17252. doi: 10.1038/s41598-019-53463-0. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Immunohistochemistry (Frozen sections). Soldevila, Ferran et al used Alexa Fluor® 488 Conjugation Kit - Lightning-Link® (ab236553) as part of examining myeloid cells in the porcine palatine tonsil. They used the kit to conjugate Alexa Fluor® 488 to anti-pig CD4α antibody for use in confocal microscopy. In situ localization of the CD14+ cells and plasmocytoid dendritic cells in porcine palatine tonsil. CD14+ cells and pDCs were localized by confocal microscopy following ethanol fixation of tonsil slices. The areas assessed included the follicle (F), the interfollicular region (IFA), the crypt (C). The tissue was stained using panel 1 antibodies; white arrow CD14+ cells and yellow arrow pDCs. Images are representative of at least two images from each section, from three different pigs. Objective used : (A) 63× oil immersion. Scale bars as shown.
Immunohistochemistry (Frozen sections). Soldevila, Ferran et al used Alexa Fluor® 488 Conjugation Kit - Lightning-Link® (ab236553) as part of examining myeloid cells in the porcine palatine tonsil. They used the kit to conjugate Alexa Fluor® 488 to anti-pig CD4α antibody for use in confocal microscopy. In situ localization of the CD14+ cells and plasmocytoid dendritic cells in porcine palatine tonsil. CD14+ cells and pDCs were localized by confocal microscopy following ethanol fixation of tonsil slices. The areas assessed included the follicle (F), the interfollicular region (IFA), the crypt (C). The tissue was stained using panel 1 antibodies; white arrow CD14+ cells and yellow arrow pDCs. Images are representative of at least two images from each section, from three different pigs. Objective used : (A) 63× oil immersion. Scale bars as shown. Image from Soldevila, Ferran et al. Frontiers in immunology vol. 9 1800. 15 Aug. 2018, doi:10.3389/fimmu.2018.01800. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Immunocytochemistry. Direct immunofluorescent staining using Recombinant Anti-EGFR antibody [E235] - BSA and Azide free (ab227459) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553). Lightning-Link® Alexa Fluor® 488 Anti-EGFR antibody [E235] conjugate was used to stain wild-type A431 cells (top panel) and MCF7 negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-EGFR Alexa Fluor® 488 conjugate (shown in green) or ab227459 (Anti-EGFR antibody, unlabelled control) overnight at +4°C. ab227459 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Immunocytochemistry. Direct immunofluorescent staining using Recombinant Anti-EGFR antibody [E235] - BSA and Azide free (ab227459) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553). Lightning-Link® Alexa Fluor® 488 Anti-EGFR antibody [E235] conjugate was used to stain wild-type A431 cells (top panel) and MCF7 negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 μg/ml of LL-Anti-EGFR Alexa Fluor® 488 conjugate (shown in green) or ab227459 (Anti-EGFR antibody, unlabelled control) overnight at +4°C. ab227459 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
ELISA. A polyclonal antibody was purchased from a commercial source and conjugated to Alexa Fluor® 488 using Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553) and to CF™488A dye using a competitor conjugation kit. The conjugates were tested by ELISA and the fluorescence measured at 523nm after excitation at 487nm. The Abcam conjugate out-performs the competitor.
ELISA. A polyclonal antibody was purchased from a commercial source and conjugated to Alexa Fluor® 488 using Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553) and to CF™488A dye using a competitor conjugation kit. The conjugates were tested by ELISA and the fluorescence measured at 523nm after excitation at 487nm. The Abcam conjugate out-performs the competitor.

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
Alexa Fluor® 488 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

ab236553 Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link®
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