各位小伙伴，您是否在蛋白免疫印記時，面對各種內參抗體而不知所措呢，內參蛋白是生物體內管家基因編碼的，在多組織中穩定表達，用于驗證免疫學實驗中是否正常進行和一種半定量檢測蛋白表達的標準對照，那么如何正確的選擇內參抗體呢！可以從以下幾點去參考。
 

一、樣本種屬來源
 

1. 哺乳動物組織或著細胞來源的樣本，一般選擇β-actin、α-tubulin、GAPDH、Lamin- B、Histone H3、Na+/K+-ATPase。

2. 植物來源樣本，一般選擇plant actin、Rubisco等。

3.其他研究較稀少的樣本，通過查詢相關文獻或數據庫，選擇合適的蛋白作為內參。

二、目的蛋白分子量
 

保證目的蛋白分子量與內參相差大于5KDa以上，防止檢測時干擾，例如，目的蛋白為40KDa，不宜選擇β-actin（42KDa）作內參，可考慮GAPDH (36KDa) 或者β-tubulin(55KDa)作為內參。
 

三、內參蛋白表達部位

因此，提取蛋白的部位不同，選擇的內參蛋白也不同。比如有時胞質、胞核分開提取而不提取總蛋白時，就要選擇相應部位表達的內參蛋白。如 H3在細胞核中高效穩定表達，理論上在細胞質不表達，那么對于胞質提取的蛋白，就不能選用H3作為內參蛋白，而應該選擇細胞質中表達的β-actin 、GAPDH α-Tubulin等作為內參蛋白。（內參評價推薦軟件geNorm、NormFinder）

注意：使用內參抗體需避開以下情況

文獻引用：

[1]Zheng G, Jiang C, Li Y, et al. TMEM43-S358L mutation enhances NF-κB-TGFβ signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy[J]. Protein & cell, 2018: 1-16.

IF（6.228）

[2] Wang T, Wang J, Wang J, et al. HAP1 is an in vivo UBE3A target that augments autophagy in a mouse model of Angelman syndrome[J]. Neurobiology of disease, 2019, 132: 104585.    30101   IF 5.36

[3] Peng RR, Wang LL, Gao WY, et al. The 5.8S pre-rRNA maturation factor, M-phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis. Cell Prolif. 2020;53(3):e12769.  

GAPDH    IF: 5.753

[4] Tan K S, Zhang Y, Liu L, et al. Molecular cloning and characterization of an atypical  butyrylcholinesterase-like protein in zebrafish[J]. Comparative Biochemistry and Physiology  Part B: Biochemistry and Molecular Biology, 2021: 110590.   Flag-Tag (DYKDDDDK), mouse  30503ES20

[5] Zhuang W, Yan Z. The S2–S3 Loop of Kv7. 4 Channels Is Essential for Calmodulin Regulation of  Channel Activation[J]. Frontiers in Physiology, 2020, 11.   30601ES20
[6] Wang Y, Hou Y, Qiu J, et al. Abscisic acid promotes jasmonic acid biosynthesis via a ‘SAPK10 ‐bZIP72‐AOC’pathway to synergistically inhibit seed germination in rice (Oryza sativa)[J].  The New Phytologist, 2020, 228(4): 1336.   GST\HIS\GFP coip

[7] S Chen, et al. Novel Role for Tranilast in Regulating NLRP 3 Ubiquitination, Vascular  Inflammation, and Atherosclerosis JAHA. June 16, 2020 Vol 9, Issue 12 αTUBULIN

[8] Wu L, Gao A, Lei Y, Li J, Mai K, Ye J. SHP1 tyrosine phosphatase gets involved in host defense  against Streptococcus agalactiae infection and BCR signaling pathway in Nile tilapia  (Oreochromis niloticus). Fish Shellfish Immunol. 2020;99:562-571.  β-actin   

[9] Wu F, Xu K, Xu K, et al. Dl-3n-butylphthalide improves traumatic brain injury recovery via  inhibiting autophagy-induced blood-brain barrier disruption and cell apoptosis. J Cell Mol  Med. 2020;24(2):1220-1232.GAPDH  

[10] Liu X , Sun T , Ge Q , et al. Construction of Novel Bispecific Single-Domain Antibodies  (BiSdAbs) with Potent Antiangiogenic Activities[J]. 2020.   GAPDH rabbit antibody