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N6-methyladenosine / m6A Rabbit mAb

概述
货号: A19841
规格: 50 μL / 100 μL / 200 μL
宿主: Rabbit
同种型: IgG
ABclonal: - N6-methyladenosine / m6A Rabbit mAb (A19841)
ABclonal: - N6-methyladenosine / m6A Rabbit mAb (A19841)
ABclonal:Immunofluorescence - N6-methyladenosine / m6A Rabbit mAb (A19841)
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背景

Discovered in the 1970s, m6A is the most prevalent internal modification in polyadenylated mRNAs and long non-coding RNAs (lncRNAs) in higher eukaryotes. m6A is widely conserved among eukaryotic species that range from yeast, plants, flies to mammals, as well as among viral RNAs with a nuclear phase. The m6A-based modification is associated with a well-defined RNA motif, RRACH (R: A/G, H: A/C/U). As a representative of the epitranscriptome, m6A mRNA modifications participate in many vital activities in the cell, including stem cell self-renewal and differentiation, mRNA transcription, alternative splicing, nuclear export, translation, degradation, and microRNA processing. These processes determine the expression or inactivation of specific genes, which is vital for growth and development.(PMID: 30416848; PMID: 24662220; PMID: 30429466)

免疫原信息

免疫原Chemical compounds corresponding to N6-methyladenosine / m6A.
序列Email for sequence
基因ID
Swiss Prot
别名N6-methyladenosine;m6A

应用

反应物种Human, Mouse, Rat, Other (Wide Range)
验证应用WBIHCICCIFIPChIPChIPseqRIPFCELISAMeDIPNucleotide ArrayDBFACSCoIP
推荐稀释比&实验步骤
  • DB   1:500 - 1:2000

  • IF   1:50 - 1:200  [ 实验步骤 ]
    • 细胞样本

      免疫荧光实验操作流程(细胞样本)

      一、实验仪器及试剂

      1、实验器材

      4℃冰箱、恒温恒湿箱、通风橱、移液器、避光孵育湿盒。

      2、实验试剂
      • (1) 缓冲液:0.01M pH7.2±0.2 TBS ;0.01M pH7.2±0.2 TBST
        试剂名称1*TBS/L试剂名称1*TBST/L
        Tris1.21g1*TBS1L
        NaCl8.8gTween 20 溶液0.001L
        用稀HCl溶液和NaOH溶液调pH值用稀HCl溶液和NaOH溶液调pH值
      • (2) 固定液:4%中性甲醛固定液(TBS缓冲液配制)
      • (3) 抗体稀释液:1X TBS / 5% 正常血清 / 0.3% Triton™ X-100
      • (4) 通透剂:0.3% Triton X-100(TBS缓冲液配制)
      • (5) 二抗(可选):
        Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L)# AS074;
        Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L)# AS073;
        Alexa Fluor 594-conjugated AffiniPure Goat Anti-Mouse IgG (H+L)# AS077;
        Alexa Fluor 488-conjugated AffiniPure Goat Anti-Mouse IgG (H+L)# AS076;
      • (6) 染核试剂:1mg/mL DAPI(用时使用0.01M pH7.2 TBS缓冲液配制)
      • (7) 去离子水(dH2O)、抗荧光衰减封片剂。

      二、实验步骤

      1.样本前处理
      • (1) 弃去培养基,在细胞上缓慢加入常温TBS,清洗2次,每次5秒;
      • (2) 细胞固定:在细胞上覆盖 4%中性甲醛固定液(TBS缓冲液配制),置于4℃,固定15min;固定液需足量;
      • (3) 固定液的清洗:去除固定液,使用4℃预冷的TBS缓冲液,漂洗3次,每次5分钟;
      注意:需避免实验操作时温差过大或温度骤变;固定液需保证足量,可过量使用;甲醛有毒性,加固定液和固定后的清洗需在通风橱内操作。
      2.染色步骤
      • (1)封闭:用5%空白山羊血清将样本完全覆盖,切片需放置于湿盒内,细胞孔板可直接将孔板密封好,置于37℃恒温恒湿培养箱孵育30min;
      • (2)一抗孵育:去除封闭液,直接在样本上滴加TBS缓冲液配制的一抗工作液,样本需完全覆盖,切片需放置于湿盒内,细胞孔板可直接将孔板密封好,置于4℃孵育过夜;
      • (3)复温:将样本置于常温,复温15min,去除抗体工作液,用缓冲液TBST洗涤1次,5分钟;用缓冲液TBS洗涤3次,每次5分钟;
      • (4)二抗孵育:在样本上滴加与一抗种属对应的荧光二抗工作液,样本需完全覆盖,避光,37℃,孵育1小时;去除二抗工作液,用缓冲液TBST洗涤1次,5分钟;用缓冲液TBS洗涤3次,每次5分钟;
      • (5)染核:在样本上滴加DAPI工作液,使用0.01M pH7.2±0.2 TBS缓冲液配制,DAPI溶液:TBS缓冲液体积比1:500,避光,室温,孵育10分钟;去除DAPI工作液,用缓冲液TBST洗涤1次,5分钟;用缓冲液TBS洗涤3次,每次5分钟;
      • (6)细胞孔板可直接加入抗荧光衰减封片剂后,于荧光显微镜下观察并采集图像;细胞涂片滴加抗荧光衰减封片剂,然后加盖盖玻片封片,再于荧光显微镜下观察并采集图像;细胞爬片可取出,盖在滴加抗荧光衰减封片剂的载玻片上后,于荧光显微镜下观察并采集图像。
      注意:实验中,所有试剂滴加应准确、快速、足量,不能出现干片的情况;染色步骤从二抗孵育开始,后续所有步骤都需注意避光操作;染色完成后需及时观察并采集图像,避免干燥和荧光物质淬灭。
    • 组织样本

      免疫荧光实验操作流程(石蜡切片)

      一、实验仪器及试剂

      1、实验器材

      微波炉、4℃冰箱、恒温恒湿箱、烘箱、移液器、避光孵育湿盒、抗原修复盒。

      2、实验试剂
      • (1) 缓冲液:0.01M pH7.2±0.2 TBS ;0.01M pH7.2±0.2 TBST
        试剂名称1*TBS/L试剂名称1*TBST/L
        Tris1.21g1*TBS1L
        NaCl8.8gTween 20 溶液0.001L
        用稀HCl溶液和NaOH溶液调pH值用稀HCl溶液和NaOH溶液调pH值
      • (2) 修复液(可选):
        pH6.0 0.01M柠檬酸修复液(1L):C6H8O7•H2O 0.4g;NaC6H5O7•2H2O 3g;pH6.0;
        pH7.0 0.01M PBS修复液(1L):KH2PO4 0.07g;NaCl 7.89g;Na2HPO4 0.45g;KCl 0.10g;pH7.2-7.4;
        pH8.0 0.05M Tris-EDTA修复液(1L):C4H11NO3 6.05g;C10H14N2Na2O8•2H2O 0.29g;用稀HCl调至pH8.0;
        pH9.0 0.01M Tris-EDTA修复液(1L):C4H11NO3 1.21g;C10H14N2Na2O8•2H2O 0.37g;pH9.0。
      • (3) 封闭液:5%空白山羊血清
      • (4) 抗体稀释液:1X TBS / 5% 正常血清 / 0.3% Triton™ X-100
      • (5) 二抗:
        Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L)# AS074;
        Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L)# AS073;
        Alexa Fluor 594-conjugated AffiniPure Goat Anti-Mouse IgG (H+L)# AS077;
        Alexa Fluor 488-conjugated AffiniPure Goat Anti-Mouse IgG (H+L)# AS076;
      • (6) 染核试剂:1mg/mL DAPI(用时使用0.01M pH7.2±0.2 TBS配制)
      • (7) 去离子水(dH2O)、抗荧光衰减封片剂。

      二、实验步骤

      1.水化/脱蜡:
      • (1) 烤片:将石蜡切片按同一朝向放置在切片架上,将其放入55℃的恒温箱中烤片30分钟;同时将脱蜡液1缸一起放入55℃的恒温箱中;
      • (2) 脱蜡至水:将石蜡切片连同切片架一起放入脱蜡液1缸中,再一起从恒温箱中取出置于常温,5分钟后,将切片取出浸入到常温脱蜡液2缸中,并按照脱蜡液2、脱蜡液3、无水乙醇1、无水乙醇2、无水乙醇3的顺序依次将石蜡切片放入缸中,每缸5分钟;用流水清洗切片5分钟。
      注意:流水清洗时水流不能直接对着切片;操作过程中需一直保持切片处于湿润状态。
      2.抗原修复:
      • (1)方法一,微波热修复:将切片浸入盛有修复液的修复盒中,将抗原修复盒盖斜盖在修复盒上;整体放入微波炉中,高火加热3分钟后停火,微波炉内静置5分钟;再高火加热3分钟后停火,微波炉内静置5分钟;随后中低火加热1分钟后停火,微波炉内静置5分钟,然后将切片连同抗原修复盒拿出缓慢冷却至室温。
        注意:修复液需完全浸没切片上组织;修复过程中严禁打开微波炉门;修复完成后不可快速冷却;修复液可根据实验需求自行换用其他类型修复液。
      • (2)方法二,高压热修复:在高压锅中,加入抗原修复液,高火预热;待修复液沸腾后将切片置于其中,并完全浸泡组织,盖好锅盖,扣上压力阀,高火继续加热;待限压阀开始转动喷气后调至中火,同时开始计时2分钟;计时结束后离开热源,自然降压后将高压锅移入冷水中缓慢冷却。待修复液温度降至室温后,用缓冲液洗涤3次,每次1min。
        注意:修复液需完全浸没切片上组织;修复过程中严禁打开仪器或中断运行程序;修复液可根据实验需求自行换用其他类型修复液。
      3.染色:
      • (1)封闭:用5%空白山羊血清将样本完全覆盖,切片需放置于湿盒内,置于37℃恒温恒湿培养箱孵育30min;
      • (2)一抗孵育:去除封闭液,直接在样本上滴加TBS缓冲液配制的一抗工作液,样本需完全覆盖,切片需放置于湿盒内,置于4℃孵育过夜;
      • (3)复温:将样本置于常温,复温15min,去除抗体工作液,用缓冲液TBST洗涤1次,5分钟;用缓冲液TBS洗涤3次,每次5分钟;
      • (4)二抗孵育:在样本上滴加与一抗种属对应的荧光二抗工作液,样本需完全覆盖,避光,37℃,孵育1小时;去除二抗工作液,用缓冲液TBST洗涤1次,5分钟;用缓冲液TBS洗涤3次,每次5分钟;
      • (5)染核:在样本上滴加DAPI工作液,使用0.01M pH7.2 TBS缓冲液配制,DAPI溶液:0.01M pH7.2 TBS缓冲液体积比1:500,避光,室温,孵育10分钟;去除DAPI工作液,用缓冲液TBST洗涤1次,5分钟;用缓冲液TBS洗涤3次,每次5分钟;
      • (6)滴加抗荧光衰减封片剂,然后加盖盖玻片封片,再于荧光显微镜下观察并采集图像。
      注意:实验中,所有试剂滴加应准确、快速、足量,不能出现干片的情况;染色步骤从二抗孵育开始,后续所有步骤都需注意避光操作;染色完成后需及时观察并采集图像,避免干燥和荧光物质淬灭。
理论分子量
实际分子量
存储缓冲液Store at -20℃. Avoid freeze / thaw cycles.
Buffer: PBS with 0.02% sodium azide,0.05% BSA,50% glycerol,pH7.3.
实验方法
阳性样本Arabidopsis
细胞定位
纯化方式Affinity purification

N6-methyladenosine / m6A Rabbit mAb 检测图片

ABclonal: - N6-methyladenosine / m6A Rabbit mAb (A19841) }

- N6-methyladenosine / m6A Rabbit mAb (A19841)

The m6A rabbit monoclonal antibody (4µg,A19841) are tested in Nucleotide Array against N6-methyladenosine (m6A) and unmodified adenosine (100pmol for each oligomer).
Oligomer 4 - N6-methyladenosine (m6A-UAACUGGACCGAAUGG-Biotin)
Oligomer 5 - N6-methyladenosine (AUAACUGG-m6A-CCGAAUGG-Biotin)
Oligomer 6 - unmodified adenosine (AUAACUGGACCGAAUGG-Biotin)
ABclonal: - N6-methyladenosine / m6A Rabbit mAb (A19841) }

- N6-methyladenosine / m6A Rabbit mAb (A19841)

The m6A rabbit monoclonal antibody (A19841) are tested in Dot Blot against N6-methyladenosine (m6A) and unmodified adenosine.
Oligomer 8 - ATAACTGG-m6A-CCGAATGG
Oligomer 9 - ATAACTGGACCGAATGG
Oligomer 10 - AAAAAAAAAAAAAAAA-biotin.
ABclonal:Immunofluorescence - N6-methyladenosine / m6A Rabbit mAb (A19841) }

Immunofluorescence - N6-methyladenosine / m6A Rabbit mAb (A19841)

U2OS cells pre-treated with BrdU were subjected UVC irradiation incubated at 37 °C for the indicated time, and stained for m6A rabbit monoclonal antibody (A19841). DAPI, 4′,6-diamidino-2-phenylindole. Global UVC irradiation exceed cytoplasmic leavel,peaking at 2 min after irradiation and diminishing over the following 8 min.

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