http://www.aun.edu.eg/molecular_biology/Protein%20workshop/WB-beginner.pdf

http://med.stanford.edu/labs/vanderijn-west/Protocols/Western%20Blot%20Protoco1_update.doc

Article Native electrophoresis and Western blot analysis (NEWeB): A ...

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/nativepage-bis-tris-gels.html

https://www.serva.de/servaWeb/www_root/documents/Blue%20Clear%20Native%20Gel%20Ver%200213_e.pdf

https://www.abcam.com/protocols/a-comparison-between-polyclonal-and-monoclonal

https://www.stjohnslabs.com/blog/monoclonal-antibodies-vs-polyclonal-antibodies-which-to-choose/

https://www.cedarlanelabs.com/Contents/Files?filePath=pdf%5CEMD%20Millipore%20-%20Further%20Considerations%20of%20Antibody%20Validation%20and%20Use%20-%20How%20to%20Avoid%20Reviewer%20Challenges%20-%20White%20Paper.pdf

For native western, read page 6, abcam protocol

http://www.aun.edu.eg/molecular_biology/Protein%20workshop/WB-beginner.pdf

Hi,

You can follow the link for the protocol provided by Bhairavi Vajaria. In addition i will like to suggest the following points:

1.You can use transfer buffer(3.03g of Tris, 14.4g of Glycine and 200ml of methanol and make it up to 1litre) and keep at 4 degree before use

2.You can also use PVDF membrane(ensure that you activate it before use by soaking the membrane in 100% methanol for 15 sec, followed by distilled water and equilibrate it in your transfer buffer(3-5min)

3.Transfer condition- use 25V and observe the current(if 55-59mA, run for 25min or 60-66mA , run for 20min).

4. avoid air bubbles between your membrane and the gel.

5. Run native-PAGE using 10% depending on your protein size @ 120V for 1hr.

I hope this few points will help you.

All the best!

Western blotting is usually performed with proteins denatured with SDS, which gives them a high negative charge (about 1 molecule of SDS bound per 3 amino acids). If you perform native PAGE, SDS is not present and the protein charge much lower. Blotting will be more difficult, and the protocol will have to be adapted to the specific case.

• The gel will have to be equillibrated with transfer buffer for long enough that the intended ionic conditions are met throughout the gel, but not so long that diffusional band spreading occurs. I'd try 1 h first. Including SDS in the transfer buffer may be a good idea to ease the protein out of the gel. Then I'd incubate at elevated temperature (say, 40 °C) to speed detergent binding to the protein. If you want to make zymograms (use enzymatic activity for staining) detergent of course is not an option.
• Composition of the transfer buffer: Towbin's transfer buffer after SDS-PAGE is in effect electrophoresis buffer with added methanol. Methanol increases small protein binding to the membrane, but prevents mobilisation of large proteins from the gel. The SDS present has, in effect, the opposite effect. doi:10.1006/abio.1993.1313 describes a semi-dry system with MeOH on the membrane and SDS on the gel side. doi:10.1016/0003-2697(86)90207-1 describes a very simple transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, with 50 µM SDS added for large proteins) that in my hands works better than Towbin's. I'd try either that or the electrophoresis buffer used (which may have to be diluted to reduce current - and hence heat - during blotting).
• Membrane: PVDF is more expensive than NC, but has a lot of advantages. Its protein binding capacity and affinity are much higher, protein slipping through the membrane is not an issue. It is mechanically stronger and will not fragment during the later incubation steps. And, if protein sequencing is intended, the cut-out band on PVDF can be used directly in gas-phase sequencers.
• Blotting direction: In SDS-PAGE all proteins are negatively charged because the charge of the bound SDS exceeds by far the charge of any amino acids in the protein. Hence, the membrane has to be on the positive side during blotting. In native electrophoresis protein net charge is determined by pI and buffer pH, and some proteins may move to the positive side, others to the negative. Hence, you need membranes on both sides of the gel. For the same reason, you need to add the protein sample to the middle of the gel initially, this requires horizontal electrophoresis.
• Blotting time and conditions will have to be optimised. You want to shoot for about 30 V, buffer concentration should be low enough that the current doesn't exceed 200 mA for a minigel. In my hands, tank blotters work better than semi-dry systems and are easier to cool. However, if you want different buffers for membrane and gel as discussed above, you are limited to semi-dry.
• Antibodies: Western-blotting after SDS-PAGE requires sequence-specific antibodies, because protein conformation is destroyed. After native electrophoresis, conformational antibodies are worth a try, but it would be a good idea to also use a sequence-specific antibody for control (how native is native electrophoresis, anyway ;-) ).

Bhairavi Vajaria Adesola Julius Tola Engelbert Buxbaum Thanks for your suggestions, I will try and get back to you.