Lightning-Link® AP 抗體標定試劑盒

Alkaline phosphatase Conjugation Kit - Lightning-Link®

貨號

ab102850

包裝

3 x 10 μg
100 μg
3 x 100 μg
1 mg

廠牌

Abcam

產品簡介

Abcam Alkaline phosphatase Conjugation Kit - Lightning-Link® 提供了一種簡便快速的方法，可將 AP 酵素（alkaline phosphatase，又稱為鹼性磷酸酶）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 AP 酵素試劑小瓶，即可啟動標定反應；動手操作時間約 30 秒，3.5 小時左右即可完成標定，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

  • 操作簡便快速，套組採用 Lightning-Link® 直接標定技術，僅需將添加有 Modifier 的待標定抗體與凍乾的 AP 酵素試劑混合，即可透過離胺酸 (lysine) 等游離胺基進行共價標定，動手操作時間僅約 30 秒，整體反應時間在 3.5 小時左右（標定反應約 3 小時＋中止反應 30 分鐘）。
  • 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。
ECL slot blot assay. Grabias B et al. used ab102850 as part of developing a rapid detection of Plasmodium falciparum infection in mosquitoes. They used the kit to conjugate Alkaline phosphatase to anti-Plasmodium falciparum circumsporozite protein antibody for use in ECL slot blot assay. Evaluation of whether conjugation of primary mAb 2A10 with alkaline phosphatase (1°-AP) enhanced sensitivity for the detection of Pfoocyst prepared from mosquito lysates when compared to the use of AP-conjugated secondary antibody (2°-AP). Detection limits were compared for each protocol by fitting the band intensities of serially diluted oocysts to a Michaelis-Menten regression curve and establishing a cutoff intensity threshold of mean + 2 SD from unfed mosquito specimens run on the same blot. Labeled primary antibody displayed overall higher band intensities across the range of oocyst dilutions examined and achieved lower limits of detection than the typical sandwich antibody format (0.009 oocyst versus 0.02 oocyst, respectively). The removal of an additional antibody incubation step also contributed to an overall shorter assay time in the newly developed slot blot protocol. Image from GrabiasB et al., PLoS One, 12(4): e0174229. Fig 3; doi: 10.1371/journal.pone.0174229. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Western Blot. Feng, Linyuan, et al used Alkaline phosphatase Conjugation Kit - Lightning-Link® (ab102850) as part of examining the interaction between UL23 and Nmi identified by coimmunoprecipitation. They used the kit to conjugate Alkaline phosphatase to primary antibodies for use in western blot. (A-B) Human U251 cells were co-transfected with a combination of two plasmids expressing FLAG- and HA-tagged proteins, and then harvested at 48 hours posttransfection. (C-D) Human U251 cells were infected with human cytomegalovirus (HCMV), a human herpesvirus, (MOI = 1) and cellular lysates were prepared at 48-72 hours postinfection. The input protein samples (80 μg) (Input) (lanes 3, 6, 9, 12, 15, 18, 21, and 24) and samples (15 μg) that were precipitated with anti-HA (IP (anti-HA)) (lanes 2, 5, 8, and 11), anti-FLAG (IP (anti-FLAG)) (lanes 1, 4, 7, and 10), anti-Nmi (lanes 13, 16, 19, and 22), anti-UL44 (lanes 14 and 17), or anti-UL23 antibodies (lanes 20 and 23), were separated on SDS-containing polyacrylamide gels, and assayed with Western blot analysis using anti-HA (anti-HA) (A), anti-FLAG (anti-FLAG) (B), anti-UL44 (anti-UL44) (C), anti-UL23 (anti-UL23) (D), and anti-Nmi (anti-Nmi) (C-D) antibodies that were directly conjugated to alkaline phosphatase using the Lightning-Link® kit ab102850, respectively. Image from Feng, Linyuan, et al., PLoS pathog., 14(1): e1006867; doi: 10.1371/journal.ppat.1006867. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Sandwich ELISA. Charlermroj R et al. used ab102850 to run a sandwich ELISA and compare its sensitivity with a microsphere immunoassay based on Luminex using the same set of antibodies. Image from Charlermroj R et al., PLoS One, 8(4): e62344. Fig 5; doi: 10.1371/journal.pone.0062344. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
Microfluidic sandwich ELISA. Thaitrong N et al. used ab102850 to run a microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. Nine different conditions for each disease panel were tested on the microfluidic platform using combinations of three concentrations of capture Ab (11E5, 2D6, and 5E7) and three concentrations of detection Ab (MPC-AP, MYSV6-AP). Image from Thaitrong N et al., PLoS One, 8(12): e83231. Fig .; doi: 10.1371/journal.pone.0083231. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
ELISA. Thomas FC et al used ab102850 as part of examining the toxicity of p17 protein assemblies. They used the kit to conjugate Alkaline phosphatase to anti-bovine Hp antibody for use in ELISA. Boxplot showing Hp concentration (μg/ml) in two SCC categories of composite milk samples * indicates an extreme value (values greater than 3 interquartile range (IQR) away from 25th or 75th percentile); IQR = 3rd quartile -1st quartile (represented by the height of the box). ° indicates an outlier value (values greater than 1.5 interquartile range (IQR) away from 25th or 75th percentile); IQR = 3rd quartile -1st quartile (represented by the height of the box). Image from Thomas FC et al., BMC Vet Res., 11, 207. Fig 1.; doi: 10.1186/s12917-015-0533-3. 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（> 1 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% BSA50% glycerol
0.1% sodium azidePBSPotassium phosphate
Sodium chlorideHEPESSucrose
Sodium citrateEDTATrehalose
1 Tris buffered saline is almost always ≤ 50 mM / 0.6%
Incompatible buffer constituents
ThiomerosalProclinGlycine
ArginineGlutathioneDTT

套組內容物

Item3 x 10 µg100 µg3 x 100 µg1 mgStorage temperature
Alkaline phosphatase 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

ab102850 Alkaline phosphatase Conjugation Kit - Lightning-Link®
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