Why is pvdf better than nitrocellulose

PLoS One. Valid application of western blotting. Mol Biol Rep. Epub Feb 8. PMID : Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PMID: Western blot: technique, theory, and trouble shooting. N Am J Med Sci. Powered by Social Snap. Copy link. Copy Copied. How protein-loving is it? Western blotting especially high molecular weight proteins Protein sequencing Amino acid analysis Solid phase assay systems.

Both nitrocellulose and PVDF come in typical pore sizes of 0. The 0. Nitrocellulose membranes have high protein-binding affinity but are not capable of the detection sensitivity of PVDF membranes. On the plus side, you may get lower background signal if using nitrocellulose. In other words, PVDF membrane shows more advantages in the ability to bind with glycans in proteins.

The intensity analysis of lectin staining of the glycoproteins bound to PVDF membrane were shown to increase 1. Similar results can also be observed in our recently published article Although the LB staining principle of glycoprotein is different from protein staining principle of IB, the more proteins remain on the membrane, the more glycan that attached to the corresponding proteins, so it is easier to be recognized by lectin. Carefully observing these stained bands, we found that the molecular weight of the stained glycoprotein was concentrated between 40 and kDa or higher when the loading amount of serum sample was 0.

The results in Figs. Clinical samples are sometimes very limited. If more than two targets can be detected after one electrophoresis, it will save samples, materials and time. Therefore, we explored the antibody re-probed ability of the two membranes in the following four cases: 1 staining with one lectin and then re-probing with another lectin; 2 staining with one antibody and then re-probing with another antibody; 3 staining with lectin and then re-probing with antibody; 4 staining with antibody and then re-probing with lectin.

Briefly, diluted healthy mixed sera proteins 3—0. After reacting with primary and secondary antibodies, the protein band was visualized by using ECL reagents. Then, membrane stripping solutions were employed to remove the antibodies that bound on the membranes, and the blotted membranes were re-blocked with BSA and re-probed with other antibodies. The results were shown in Fig. This is consistent with the result of Fig.

The pooled sera proteins 3. In the results we mentioned above, we detected the binding ability of protein or glycoprotein to PVDF and NC membranes by incubating proteins which were transferred to PVDF and NC membranes, and compared the antibody re-probed ability of the two membranes.

However, uneven electric transfer efficiency and different degrees of protein loss existed in electric transfer In order to further confirm the above results, we directly incubate four proteins with different molecular weights onto activated PVDF and NC membranes, respectively, so as to detect the direct binding ability of the two membranes to proteins. The results of signal intensity analysis of the staining blots were shown in Fig.

The results of statistical analysis showed that under any concentration, the protein directly incubated onto NC membrane had better binding ability than that incubated onto PVDF membrane, which is consistent with the result in Fig.

The left side of a, b, c, and d: representative blots; The right side of a — d the relative signal intensity of the proteins that binded on the two membranes were detected by staining with DB The molecular weight of fetuin is 40 kDa, although the binding ability of fetuin to NC increased gradually with the increase of fetuin concentration 40 kDa, Fig. These data are consistent with the result in Fig. In addition, the direct binding ability of the two membranes to high molecular proteins such as lactase kDa, Fig.

As shown in Fig. The results of statistical analysis of the stained membranes showed that PVDF membrane has higher lactase binding capacity than NC membrane, which is consistent with the result in Fig.

In this study, by incubating proteins which were transferred to PVDF or NC membranes with a series of antibodies and different types of lectins, we investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein. Moreover, in order to avoid the error caused by the losses of proteins during electrotransfer, we also verified the above results by directly incubating proteins having different molecular weights onto PVDF or NC membranes.

These results showed that the binding ability of NC membrane to low molecular weight protein is better than that of PVDF membrane. While PVDF membrane shows more advantages in the ability to bind to high molecular weight proteins and glycoproteins. As for the binding ability to medium molecular weight protein, there is no difference between these two membranes. In addition, we also explored the transfer time for different antigens Table 2 , such as ApoA1 50 min, EEF1A2 70 min, CerP 90 min, other antigens and glycoprotein 60 min.

Therefore, to improve the detection sensitivity of protein in WB, we should select the solid phase supporter according to the molecular weight of the target protein, and simultaneously also consider the corresponding transfer time.

When the molecular weight of protein is low, the membrane transfer time can be appropriately shortened, otherwise, it can be appropriately extended, so as to retain more proteins on electroblotted membranes. The pore size of the membrane will also affect the detection efficiency of protein in WB. The pore size used in this study is 0. It is reported that when the molecular weight of protein is lower than 20 kD, it is more suitable to use 0. Then, for a molecular weight less than 20 kD protein, whether its binding ability to PVDF membrane and NC membrane is consistent with that of 0.

S1 a, left , but both of them were not as good as that of protein to 0. S1 a, right , which was consistent with the conclusion of 0. WB technique is largely used for the identification of proteins and the characterization of their biological functions. Its key steps, such as the selection of the solid phase supporter, transfer time and the molecular weight of the target protein can affect the sensibility and reproducibility of this technique.

Especially when the target protein is a low abundance protein, improper selection of membrane will cause difficulty in detection, or even the protein cannot be detected. Since WB is a multistep protocol, the variations of any step will affect the final detection result of protein in this technique.

Such as buffer composition, incubation times of primary antibody, contaminating chemicals, and different ECL detection reagents etc. Further studies should explore and define the optimal WB conditions through a comprehensive evaluation of a variety of factors.

Ubiquitous importance of protein glycosylation. Methods Mol. Article Google Scholar. Zuo, W. Dual characteristics of novel HER2 kinase domain mutations in response to HER2-targeted therapies in human breast cancer. Cancer Res. Yildirim, A. Relationship between serum levels of some acute-phase proteins and stage of disease and performance status in patients with lung cancer. MathSciNet Google Scholar. Zhang, Y. AGP1 acts as a biomarker for diagnosis of laryngeal cancer. Towbin, H. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.

And how do you choose which one to use in your application? To do this, you need to take the membrane type, pore size and membrane format into consideration to help you choose which membrane to use.

Protein binding capacity. Since PVDF has a higher protein binding capacity, it also offers higher sensitivity. While this feature allows it to detect lowly expressed proteins, you are more likely to get higher background noise in your antibody detection steps when using this membrane. Nitrocellulose membrane, on the other hand, may not be capable of the detection sensitivity of PVDF membranes but it will produce a lower background noise. Binding interactions. Protein molecules bind to nitrocellulose membranes through hydrophobic interactions while molecules bind to PVDF membranes through hydrophobic and dipole interactions.

However, please note that nitrocellulose requires the use of methanol in the transfer buffer which may reduce the pore size of the gel and cause high molecular weight proteins to precipitate. Physical characteristics.

While nitrocellulose is brittle and fragile, PVDF is more durable and has higher chemical resistance making it ideal for reprobing and sequencing applications.