敲除驗(yàn)證抗體
為什么要對(duì)抗體進(jìn)行敲除驗(yàn)證？
找零難？支付寶應(yīng)運(yùn)而生了！ 打車難？滴滴出行應(yīng)運(yùn)而生了！ 抗體特異性驗(yàn)證難？敲除驗(yàn)證應(yīng)運(yùn)而生了！
敲除是怎么做的？
具備CRISPR/Cas9技術(shù)平臺(tái)和慢病毒/腺病毒轉(zhuǎn)染平臺(tái)。
下圖為慢病毒侵染系統(tǒng)和CRISPR/Cas9技術(shù)結(jié)合轉(zhuǎn)染目的細(xì)胞。
抗體是怎么進(jìn)行敲除驗(yàn)證的？
以BECN1為例：
（1）選擇序列并化學(xué)合成20nt的guide序列及其互補(bǔ)序列，構(gòu)建含有sgRNA序列信息的LentiCRISPRv2重組載體。
（2）重組載體和慢病毒包裝質(zhì)粒共同轉(zhuǎn)染293T細(xì)胞，收集病毒顆粒直接侵染MCF-7細(xì)胞株，通過嘌呤霉素篩選，最終獲取穩(wěn)定轉(zhuǎn)染的細(xì)胞株。
（3）在細(xì)胞株內(nèi)，Cas9和sgRNA結(jié)合成剪切復(fù)合體，被轉(zhuǎn)運(yùn)進(jìn)入細(xì)胞核，以完成對(duì)sgRNA互補(bǔ)DNA片段的剪切。測序結(jié)果如下：
（4）抗體的敲除驗(yàn)證結(jié)果：
利用成功敲除BECN1的細(xì)胞株檢測相應(yīng)的抗體，結(jié)果顯示敲除成功的細(xì)胞系中，抗體檢測到的BECN1表達(dá)量低。證明這些抗體是特異識(shí)別目的蛋白的。檢測結(jié)果如下：
Cloud-Clone部分抗體的敲除驗(yàn)證結(jié)果
Cloud-Clone經(jīng)過敲除驗(yàn)證的部分抗體列表
| 貨號(hào) | 指標(biāo) | 敲除驗(yàn)證使用的細(xì)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 |