Description
Factor VIII Paired Antibody Set
Affinity’s Factor VIII Paired Antibody Set consists of matched capture and detecting antibodies that have been titrated and optimized for use in sandwich style ELISA assays. The product as provided contains sufficient capture and detecting antibodies for four full 96-well microplates and contains a detailed protocol sheet containing directions for use, recipes for solutions and sources for additional materials required. This Factor VIII Paired Antibody Set is intended to facilitate the end user in establishing an “in-house” immunoassay for research purposes only and must not be used for diagnostic applications. Assay validation is the responsibility of the end user.
Product Code: F8C-EIA
Supplied Materials:
- Capture Antibody (F8C-EIA-C): One yellow-capped vial containing 0.4 ml of polyclonal affinity purified anti-Factor VIII antibody for coating plates.
- Detecting Antibody (F8C-EIA-D): One red-capped tube containing 0.4 ml of peroxidase conjugated affinity-purified polyclonal anti-Factor VIII antibody for detection of captured Factor FVIII.
Related Products: VisuLize FVIII Antigen Kit Factor VIII Deficient Plasma
Species Cross Reactivity: View Chart
Product Datasheet: Factor VIII (F8 FVIII) Antigen Matched Pair Antibody Set for ELISA - F8C-EIA
Description of Factor VIII (FVIII)
Factor VIII (formerly referred to as antihemophilic globulin and Factor VIII:C) is a large glycoprotein (320 kDa) that circulates in plasma at approximately 200 ng/ml. Synthesized in the liver, the majority of Factor VIII is cleaved during expression, resulting in a heterogeneous mixture of partially cleaved forms of FVIII ranging in size from 200-280 kDa. The FVIII is stabilized by association with von Willebrand Factor to form a FVIII-vWF complex required for the normal survival of FVIII in vivo (t1/2 of 8-12 hours).
F.VIII is a pro-cofactor that is activated through limited proteolysis by thrombin. In this process F.VIIIa dissociates from vWF to combine with activated Factor IX, calcium and a phospholipid surface where it is an essential cofactor in the assembly of the Factor X activator complex. Once dissociated from vWF, FVIIIa is susceptible to inactivation by activated Protein C and by non-enzymatic decay.
Hemophilia A is a congenital bleeding disorder resulting from an X-chromosome-linked deficiency of FVIII. The severity of the deficiency generally correlates with the severity of the disease. Some Hemophiliacs (~10%) produce a FVIII protein that is partially or totally inactive. The production of neutralizing antibodies to FVIII also occurs in 5-20% of Hemophiliacs 1-3.
References and Reviews:
- Lollar P, Fay PJ, Fass DN; Factor VIII and Factor VIIIa. Methods in Enzymology, 222, pg 122, 1993.
- Hoyer, LW, Wyshock EG, Colman RW, in Hemostasis and Thrombosis, 3rd Edition, eds. RW Colman, J Hirsh, VJ Marder and EW Salzman, pp. 109-133, J.B. Lippincott Co., Philadelphia, 1994.
- Pittman DD, Kaufman RJ. Structure-Function Relationships of Factor VIII Elucidated through Recombinant DNA Technology. Thromb. Haemostas. 61:161-165, 1989.
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因为内参有好几种，分子量差别也比较大，更换分子量与检测蛋白差距更大的内参就可以避免这个问题
另外也可以先用一抗孵育显色和检测，再用Strip缓冲液洗掉膜上的抗体，重新进行内参的抗体孵育显色检测。这样也可以将检测蛋白和内参显色在同一张膜上
各位大神，有谁做WB，选用球虫的GADPH作为内参？知道哪个公司有针对球虫内参的抗体？
虽然没有具体用过检测这个抗原的抗体，但是如果希望抗体特异性好点，能检测丰度低的蛋白，还是选Abcam吧，Santa有时候全凭人品了，内参抗体什么的还能凑活，其他的比较勉强。
内参抗体：
Beta-Actin mAb (1C7) 细胞总蛋白 42 kD Abbkine A01010 小鼠源单克隆
Beta-Actin mAb (1C7) , HRP 细胞总蛋白 42 kD Abbkine A01015 小鼠源单克隆,HRP偶联
GAPDH mAb (2B5) 细胞总蛋白 36 kD Abbkine A01020 小鼠源单克隆
GAPDH mAb (2B5) , HRP 细胞总蛋白 36 kD Abbkine A01025 小鼠源单克隆,HRP偶联
Beta-Tubuline mAb (3G6) 细胞总蛋白 55 kD Abbkine A01030 小鼠源单克隆
Plant Actin mAb (3T3) 植物细胞总蛋白 42 kD Abbkine A01050 小鼠源单克隆
PCNA mAb (1D7) 细胞核蛋白 28 kD Abbkine A01040 小鼠源单克隆
COX IV mAb (14Y2) 细胞线粒体总蛋白 16 kD Abbkine A01060 小鼠源单克隆
Histone H3 mAb (2D10) 细胞核蛋白 18 kD Abbkine A01070 小鼠源单克隆
标签抗体（Tag Antibody）可用于检测各种商品化表达载体上的标签序列（如：Myc、Flag、His、GST、HA等），籍以分析目的蛋白的表达含量及其功能；
标签抗体：
Anti-Biotin Antibodies
Anti-Dye Antibodies
Anti-FITC Antibodies
Anti-Fluorescent Protein Antibodies
Anti-HRP Antibodies
Beta Galatosidase Antibodies
FLAG Tag Antibodies
GST Tag Antibodies
HA Tag Antibodies
His Tag Antibodies
Myc Tag Antibodies
TAP Tag Antibodies
V5 Tag Antibodies
一.样本种属来源：
首先要考虑的就是实验样本来源于什么物种。
1、哺乳动物的组织或者细胞样本，通常选择β-actin、β-tubulin、GAPDH、Lamin B、Histone H3、Na,K atpase等。
2、植物来源实验样本，则可以选择plantactin、Rubisco等。
3、其他来源样本研究较少，所以就应该参照文献报导，选择合适的蛋白作为内参。
二.目的蛋白分子量：
选择内参抗体时，应该考虑目的蛋白分子量的大小。通常应该保证目的蛋白与内参蛋白分子量相差5KD以上。比如目的蛋白分子量为45KD，此时不适宜选择β-actin作为内参，可以考虑选择GAPDH或者β-tubulin作为内参。
三.目的蛋白表达部位：
就一般的蛋白检测来说，β-actin、β-Tubulin抗体等就可以了，而针对于核蛋白的定量，特别是样本蛋白就是核蛋白时，选择恰当的核蛋白内参则更能体现内部参照的价值。常用的核内参抗体有Lamin A、Lamin B、Histone H3，除此之外，其它常见的核蛋白内参还有PCNA、K70、K80等，在一些文献报道中，Erk2、TATA binding protein(TBP)以及c-Jun、c-Fos等都有使用。而对于膜蛋白检测，常用的内参抗体为Na,K ATPase。对于线粒体蛋白的检测，常用VDAC1和COX IV作为内参抗体。
以上几条原则只是针对通常情况，但是需要注意的问题是——内参的选择需要考虑实际的试验环境，比如某些细胞中，由于组织缺氧、糖尿病等因素会导致GAPDH的表达增高，不适合做内参。比如在涉及细胞增殖相关试验中，c-Jun由于自身表达变化就不适合做内参;而在凋亡实验时，TBP、Lamin等也不适合作为内参。因此设计实验方案的时候应该考虑这些因素并查询相应文献，在实验过程中也应该注意如果内参表达出现异常，应考虑这方面因素。
