WesternBlot时该选择NC膜还是PVDF膜

1、Western Blot 时该选择NC膜还是 PVDF 膜？Western Blot时最常用的两种膜是硝酸纤维素膜(nitrocellulose , NC膜)和 PVDFH (又称 Positively charged nylon ) ( Polyvinylidene fluoride ,聚偏二氟乙烯膜).这两种膜各自有什么特点？我们的实验中该选择哪一种呢？来看 下面的文字.(摘自?Making and using Antibodies ?)A study of the performance of nitrocellulose, mixed ester, nylon, and covalen

2、t-binding PVDF memberanes after passive protein adsorption and also after electrotransfer was done with several different proteins labeled with125Iodine. Themembranes exhibited different binding capacities in passive adsorption tests with labeled bovine serum albumin. The PVDF showed the least, and

3、the regenerated cellulose and nylon membranes showed the most protein binding. Nitrocellulose and mixed ester membranes were midway between. In tests measuring protein retention, PVDF retained the most bound protein when washed with detergents or 5% skimmed milk. All the membranes showed virtually t

4、he same binding capacity as measured by autoradiography when tested under electrotransfer conditions with Towbin's buffer. In passive adsorption tests, the membranes wxhibited a broad range of capacities but gave similar results in electrotransfer tests.These differences were ascribed to active

5、migration of protein into the membrane matrix instead of simple diffusion and the increased hydrophobicity of Towbin's transfer buffer because of the inclusion of methanol.上面这段文字指出在被动扩散转移蛋白时,几种膜之间结合蛋白 的水平差异明显；但是 当使用转膜仪转移蛋白时,各种膜之间的差异 就很小了.The choice of membrane used for Western Blot is more criti

6、cal if the blotted protein must maintain its native conformation for detection by the antibody. For example, a comparison using a guanosine triphosphate(GTP)-overlay assay showed that the activity of a bovine GTP-binding protein was barely detectable after transfer to hydrophobic PVDF membranes but

7、was clearly detected after transfer to nitrocellulose. Western blot analysis showed the GTP-binding protein to be present on both PVDF and nitrocellulose membranes, with slightly more detected on the PVDF membranes. The authors speculated that the poor performance of PVDF in the GTP-overlay assay ma

8、y have been due to an inability of GTP-binding protein, thus immobilized, to renature correctly. Therefore, nitrocellulose might be preferred for a Western blot procedure, in which detection requires that the transferred protein regain its native conformation after transfer, such as when the blottin

9、g agent recognized threedimensional structure; for example, an antigenic epitope consisting of noncontiguous residues.由此可见,如果你的抗原表位需要维持其三维结构才能被抗体识 别,就应该优先选择NC膜.另外,Abcam网站上技术资料中的建议是：Two types of membranes are available: nitrocellulose and PVDF. The choice is personal and both work very well. PVDF mem

10、branes require careful pre-treatment: cut the membrane to the appropriate size then soak it in methanol for 1-2 min. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring,

11、 and a distorted pattern of transfer.Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich.可见,使用PVDF1时,一定要先用无水甲醇预处理,再在 transfer buffer中平衡好才可以使

12、用 PVDFM用 甲醇泡的 目 的是为了活化PVD瞋上面的正电基团,使它更容易跟带负电的蛋白质结 合.经过预处理的PVDF1在转膜时,可以使用不含甲醇的 transfer buffer .而使用NC膜时,有的需要用无水甲醇处理,有的 那么不必,直接用transfer buffer平衡好就可以了.注：我使用的 是Pall公司的NC膜,不需要无水甲醇处理,其他公司的不是很清 楚,最好参考产品说明提醒使用PVDF膜的朋友们注意两点：1. Because of the high number of protein-binding sites in the activated nylon, the ba

13、ckgrouds are normally considerably worse, but careful blocking will eliminate many of these problems.2. Chicken antibodies tend to bind PVDF and other nylonbased membranes, leading to high backgroud. Switching to a nitrocellulose membrane should help reduce background staining.通过上面的分析,根本可以得出结论,NC膜比P

14、VD瞋更通用一些.尽管NC膜能满足绝大多数情况下的要求,大家在使用NC膜时也要注意到NC膜的缺乏之处.Nitrocellulose is the most commonly used, and it or more recently developed derivatives are highly recommended. However, nitrocellulose does have certain disadvantages. The proteins are not covalently bound, and nitrocellulose can be brittle, especi

15、ally when dry. With appropriate care, however, it will fit most applications.下面这段话来自互联网,仅供参考.硝酸纤维素膜：硝酸纤维素膜是蛋白印迹实验的标准固相支持 物.在低离子转移缓冲液的环境下,大多数带负电荷的蛋白质会与硝 酸纤维素膜发生疏水作用而高亲和力的结合在一起,虽然这其中的机制还不是十分清楚,但由于硝酸纤维素膜的这个特性,而且易于封闭 非特异性结合,从而得到了广泛的应用.在非离子型的去污剂作用 下,结合的蛋白还可以被洗脱下来.根据被转移的蛋白分子量大小, 要选择不同孔径的硝酸纤维素膜.由于随着膜孔径的不断减

16、小,膜对 低分子量蛋白的结合就越牢固.但是膜孔径如果小于0.1mm蛋白的转移就很难进行了.因此,我们通常用0.45仙m和0.2仙m两种规格的硝酸纤维素膜.大于20kD的蛋白就可以用0.45 nm的膜,小于 20kD的蛋白就要用0.2仙m的膜了,如果用0.45仙m的膜就会发生 flowthrough 的现象.从膜的质地上来看,最重要的指标就是单位面积上能够结合的蛋白的量.硝酸纤维素膜的结合水平主要与膜的 硝酸纤维素的纯度有关,市场上有些硝酸纤维素膜通常会还有大量的 醋酸纤维素,因而降低了蛋白的结合量.如果采用的是100%屯度的硝一 ,一 .一2.酸纤维素,保证了最大的蛋白结合量,可达 80-150仙g/cm.由于 100%勺纯度,因而也大大减少了非特异性的结合,降低杂交背景,无 需高严谨度的洗脱步骤.其次,膜的强度和韧性也是需要考虑的因 素.常规的硝酸纤维素膜比拟脆,漂洗一两次就会破损,不能反复使 用.PVD限移膜：PVD呢一种高强度、耐腐蚀的物质,通常是用来制 造水管的.PVD