WesternBlot時(shí)*常用的兩種膜是硝酸纖維素膜（nitrocellulose，NC膜）和PVDF膜（又稱Positively chargednylon）（Polyvinylidene fluoride，聚偏二氟乙烯膜）。
這兩種膜各自有什么特點(diǎn)？我們的實(shí)驗(yàn)中該選擇哪一種呢？來(lái)看下面的文字。（摘自《Making and usingAntibodies》）
A study of the performance of nitrocellulose,mixed ester, nylon, and covalent-binding PVDF memberanes afterpassive protein adsorption and also after electrotransfer was donewith several different proteins labeled with 125Iodine.The membranes exhibited different binding capacities in passiveadsorption tests with labeled bovine serum albumin. The PVDF showedthe least, and the regenerated cellulose and nylon membranes showedthe most protein binding. Nitrocellulose and mixed ester membraneswere midway between. In tests measuring protein retention, PVDFretained the most bound protein when washed with detergents or 5%skimmed milk. All the membranes showed virtually the samebinding capacity as measured by autoradiography when tested underelectrotransfer conditions with Towbin's buffer. In passiveadsorption tests, the membranes wxhibited a broad range ofcapacities but gave similar results in electrotransfer tests. Thesedifferences were ascribed to active migration of protein into themembrane matrix instead of simple diffusion and the increasedhydrophobicity of Towbin's transfer buffer because of the inclusionof methanol.
上面這段文字指出在被動(dòng)擴(kuò)散轉(zhuǎn)移蛋白時(shí)，幾種膜之間結(jié)合蛋白的能力差別明顯；但是當(dāng)使用轉(zhuǎn)膜儀轉(zhuǎn)移蛋白時(shí)，各種膜之間的差別就很小了。
The choice of membrane used for Western Blot ismore critical if the blotted protein must maintain its nativeconformation for detection by the antibody. For example, acomparison using a guanosine triphosphate(GTP)-overlay assay showedthat the activity of a bovine GTP-binding protein was barelydetectable after transfer to hydrophobic PVDF membranes but wasclearly detected after transfer to nitrocellulose. Western blotanalysis showed the GTP-binding protein to be present on both PVDFand nitrocellulose membranes, with slightly more detected on thePVDF membranes. The authors speculated that the poor performance ofPVDF in the GTP-overlay assay may have been due to an inability ofGTP-binding protein, thus immobilized, to renature correctly.Therefore, nitrocellulose might be preferred for a Western blotprocedure, in which detection requires that the transferred proteinregain its native conformation after transfer, such as when theblotting agent recognized three dimensional structure; for example,an antigenic epitope consisting of noncontiguous residues.
由此可見，如果你的抗原表位需要維持其三維結(jié)構(gòu)才能被抗體識(shí)別，就應(yīng)該優(yōu)先選擇NC膜。
另外，Abcam網(wǎng)站上技術(shù)資料中的建議是：
Two types of membranes are available:nitrocellulose and PVDF. The choice is personal and both work verywell. PVDF membranes require careful pre-treatment: cut themembrane to the appropriate size then soak it in methanol for 1-2min. Incubate in ice cold transfer buffer for 5 minutes. The gelneeds to equilibrate for 3-5 minutes in ice cold transfer buffer.Failure to do so will cause shrinking while transferring, and adistorted pattern of transfer.
Methanol is only necessary if usingnitrocellulose. If using PVDF, methanol can be removed from thetransfer buffer altogether, and is only needed to activate thePVDF before assembling the gel/membrane sandwich.可見，使用PVDF膜時(shí)，一定要先用無(wú)水甲醇預(yù)處理，再在transferbuffer中平衡好才可以使用（PVDF膜用甲醇泡的目的是為了活化PVDF膜上面的正電基團(tuán)，使它更容易跟帶負(fù)電的蛋白質(zhì)結(jié)合）。經(jīng)過(guò)預(yù)處理的PVDF膜在轉(zhuǎn)膜時(shí)，可以使用不含甲醇的transferbuffer。而使用NC膜時(shí)，有的需要用無(wú)水甲醇處理，有的則不必，直接用transferbuffer平衡好就可以了。（注：我使用的是Pall公司的NC膜，不需要無(wú)水甲醇處理，其他公司的不是很清楚，*好參考產(chǎn)品說(shuō)明）
提醒使用PVDF膜的朋友們注意兩點(diǎn)：
1. Because of thehigh number of protein-binding sites in the activated nylon,the backgrouds are normally considerably worse, but carefulblocking will eliminate many of these problems.
2. Chicken antibodies tend tobind PVDF and other nylon-based membranes, leading to highbackgroud. Switching to a nitrocellulose membrane should helpreduce background staining.
通過(guò)上面的分析，基本可以得出結(jié)論，NC膜比PVDF膜更通用一些。盡管NC膜能滿足絕大多數(shù)情況下的要求，大家在使用NC膜時(shí)也要注意到NC膜的不足之處。
Nitrocellulose is the most commonly used, and itor more recently developed derivatives are highly recommended.However, nitrocellulose does have certain disadvantages. Theproteins are not covalently bound, and nitrocellulose can bebrittle, especially when dry. With appropriate care, however, itwill fit most applications.
下面這段話來(lái)自互聯(lián)網(wǎng)，僅供參考。
硝酸纖維素膜：硝酸纖維素膜是蛋白印跡實(shí)驗(yàn)的標(biāo)準(zhǔn)固相支持物。在低離子轉(zhuǎn)移緩沖液的環(huán)境下，大多數(shù)帶負(fù)電荷的蛋白質(zhì)會(huì)與硝酸纖維素膜發(fā)生疏水作用而高親和力的結(jié)合在一起，雖然這其中的機(jī)制還不是十分清楚，但由于硝酸纖維素膜的這個(gè)特性，而且易于封閉非特異性結(jié)合，從而得到了廣泛的應(yīng)用。在非離子型的去污劑作用下，結(jié)合的蛋白還可以被洗脫下來(lái)。根據(jù)被轉(zhuǎn)移的蛋白分子量大小，要選擇不同孔徑的硝酸纖維素膜。因?yàn)殡S著膜孔徑的不斷減小，膜對(duì)低分子量蛋白的結(jié)合就越牢固。但是膜孔徑如果小于0.1mm，蛋白的轉(zhuǎn)移就很難進(jìn)行了。因此，我們通常用0.45μm和0.2μm兩種規(guī)格的硝酸纖維素膜。大于20kD的蛋白就可以用0.45μm的膜，小于20kD的蛋白就要用0.2μm的膜了，如果用0.45μm的膜就會(huì)發(fā)生“Blowthrough”的現(xiàn)象。從膜的質(zhì)地上來(lái)看，*重要的指標(biāo)就是單位面積上能夠結(jié)合的蛋白的量。硝酸纖維素膜的結(jié)合能力主要與膜的硝酸纖維素的純度有關(guān)，市場(chǎng)上有些硝酸纖維素膜通常會(huì)還有大量的醋酸纖維素，因而降低了蛋白的結(jié)合量。如果采用的是100%純度的硝酸纖維素，保證了*大的蛋白結(jié)合量，可達(dá)80-150μg/cm2。由于100%的純度，因而也大大減少了非特異性的結(jié)合，降低雜交背景，無(wú)需高嚴(yán)謹(jǐn)度的洗脫步驟。其次，膜的強(qiáng)度和韌性也是需要考慮的因素。常規(guī)的硝酸纖維素膜比較脆，漂洗一兩次就會(huì)破損，不能反復(fù)使用。
PVDF轉(zhuǎn)移膜：PVDF是一種高強(qiáng)度、耐腐蝕的物質(zhì)，通常是用來(lái)制造水管的。PVDF膜可以結(jié)合蛋白質(zhì)，而且可以分離小片段的蛋白質(zhì)，*初是將它用于蛋白質(zhì)的序列測(cè)定，因?yàn)橄跛崂w維素膜在Edman試劑中會(huì)降解，所以就尋找了PDVF作為替代品，雖然PDVF膜結(jié)合蛋白的效率沒有硝酸纖維素膜高，但由于它的穩(wěn)定、耐腐蝕使它成為蛋白測(cè)序理想的用品，一直沿用至今。PVDF膜與硝酸纖維素膜一樣，可以進(jìn)行各種染色和化學(xué)發(fā)光檢測(cè)，也有很廣的適用范圍。這種PVDF膜，靈敏度、分辨率和蛋白親和力在精細(xì)工藝下比常規(guī)的膜都要高，非常適合于低分子量蛋白的檢測(cè)。但PVDF膜在使用之前必需用純甲醇進(jìn)行浸泡飽和1-5秒鐘。
----------轉(zhuǎn)載自lunar sea