敲除验证抗体
为什么要对抗体进行敲除验证?
找零难?支付宝应运而生了! 打车难?滴滴出行应运而生了! 抗体特异性验证难?敲除验证应运而生了!
敲除是怎么做的?
具备CRISPR/Cas9技术平台和慢病毒/腺病毒转染平台。 下图为慢病毒侵染系统和CRISPR/Cas9技术结合转染目的细胞。
抗体是怎么进行敲除验证的?
以BECN1为例:
(1)选择序列并化学合成20nt的guide序列及其互补序列,构建含有sgRNA序列信息的LentiCRISPRv2重组载体。
(2)重组载体和慢病毒包装质粒共同转染293T细胞,收集病毒颗粒直接侵染MCF-7细胞株,通过嘌呤霉素筛选,最终获取稳定转染的细胞株。
(3)在细胞株内,Cas9和sgRNA结合成剪切复合体,被转运进入细胞核,以完成对sgRNA互补DNA片段的剪切。测序结果如下:
(4)抗体的敲除验证结果:
利用成功敲除BECN1的细胞株检测相应的抗体,结果显示敲除成功的细胞系中,抗体检测到的BECN1表达量低。证明这些抗体是特异识别目的蛋白的。检测结果如下:
Cloud-Clone部分抗体的敲除验证结果
Cloud-Clone经过敲除验证的部分抗体列表
货号 | 指标 | 敲除验证使用的细胞系 |
---|---|---|
MAA788Hu22 | Programmed Cell Death Protein 1 Ligand 1 (PDCD1LG1) | MB231 |
MAB062Hu22 | Heat Shock 70kDa Protein 1 Like Protein (HSPA1L) | Raji, Hela, HepG2 |
MAC823Hu22 | Thymidine Kinase 1, Soluble (TK1) | MCF7, 293T, Hela |
MAE786Hu21 | Annexin A3 (ANXA3) | HepG2 |
MAE787Hu22 | Annexin A1 (ANXA1) | Hela, HepG2 |
PAA009Hu02 | Angiopoietin 2 (ANGPT2) | K562 |
PAA150Hu01 | Carcinoembryonic Antigen (CEA) | Hela |
PAA153Hu01 | Alpha-Fetoprotein (aFP) | HepG2 |
PAA492Hu01 | Keratin 1 (KRT1) | A431, A375, H460, Hela |
PAA537Hu02 | Enolase, Neuron Specific (NSE) | HepG2, 293T, A549, MCF7 |
PAA651Hu01 | Hyaluronan Binding Protein 1 (HABP1) | Hela, A549 |
PAA751Hu01 | Programmed Cell Death Protein 1 (PDCD1) | Jurkat |
PAA778Hu01 | B-Cell Leukemia/Lymphoma 2 (Bcl2) | Jurkat, U-937 |
PAA853Hu03 | Caspase 8 (CASP8) | Hela, HepG2 |
PAB215Hu03 | Fibrinogen Beta (FGb) | Hela |
PAB231Hu01 | Keratin 18 (KRT18) | Hela, MCF7 |
PAB233Hu01 | Cyclin Dependent Kinase 4 (CDK4) | Hela, MCF7 |
PAB698Hu01 | Lactate Dehydrogenase B (LDHB) | Jurkat, PC3, A431 |
PAB769Hu01 | S100 Calcium Binding Protein A6 (S100A6) | Hela, A549, MCF7 |
PAB905Hu01 | Interleukin 7 Receptor (IL7R) | K562, Jurkat |
PAC598Hu03 | Vanin 1 (VNN1) | BXPC-3, PC-3 |
PAE528Hu01 | Phosphoglycerate Mutase 1, Brain (PGAM1) | A431 |
PAE748Hu01 | Karyopherin Alpha 2 (KPNa2) | HepG2, 293T, Raji |
PAF460Hu01 | Folate Receptor 1, Adult (FOLR1) | Hela, MCF7 |
PAA399Mu01 | High Mobility Group Protein 1 (HMG1) | HepG2 |
PAA700Mu01 | Membrane Protein, Palmitoylated 6 (MPP6) | HepG2, 293T |
PAK078Mu01 | Fas Associating Death Domain Containing Protein (FADD) | RaW264.7 |
PAA849Ra01 | Glycogen Phosphorylase, Liver (PYGL) | HepG2 |
PAB083Ra01 | Superoxide Dismutase 2, Mitochondrial (SOD2) | Hela, MCF7 |
PAA929Bo01 | Retinol Binding Protein 4, Plasma (RBP4) | HepG2 |