Dear All,
I would like to detect NFAT (130-160kda) and its loading control histone h3 (15kda) together on same membrane. However I have never transfered more than 100 kda protein before. Many WB guides in internet states some optimal condition for either large or small protein transfer, but not sure what is best for transfering them together.
I usually use PVDF and Towbin buffer with 20% methanol as transfer buffer and transfering condition is 100V or 50mA for 1hr in 4 degree C. Should I reduce %methanol or add SDS in transfer buffer and change the transfering condition to overnight with lower current/voltage for transfer large and small protein together?
Thank you very much
Cheerio,
Hi Asa,
Your protocol is fine for both small and large proteins. But you wish to extract histone proteins, now that is very difficult. The gradient from too large (15-160 kda) for you to get clear resolution, unless both your antibodies for NFAT and H3 are good running a 12-15% gel results may be little tricky.
Assuming both the antibodies are good extract the proteins (total protein) using this protocol http://www.cell.com/developmental-cell/abstract/S1534-5807(04)00367-3?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1534580704003673%3Fshowall%3Dtrue, and then run total protein on 12-15 % SDS PAGE and keep all the things same as before. Good luck.
Regards,
Prasad.
Hi Asa,
I used to have problem with Western and tested many of the conditions you describe (MeOH, SDS, time, membrane type) systematically and I did not see any significant difference in transfer yields. If you do not want a very good quantification with WB, but only to detect if the protein is present (like a difference between "massive", "normal", "decreased" and "none"), I suggest you don't bother with optimisation.
In my case (I had a set of membrane proteins, ranging from 20 to 180 kDa), optimisation of primary and secondary incubation times and concentrations was much more fruitful than that of transfer conditions.
Hope it helps, and good luck!
Kamil
I work with histone proteins and what I usually do is to incubate the PVDF membrane in 100% methanol prior transfer, and then use Towbin without methanol and without SDS for the transfer. The voltage is fine (I use 100V or 50-110 mA), but transfer of histones require a longer time, around 8-16 hours at 4°C, possibly with stirring of the buffer, if you have the adequate transfer chamber. Given the large gap of molecular weight among your proteins, what I would suggest you to do is to try a gradient gel to run your SDS-PAGE (let's say 4-20% acrylamide gradient) if you have the chance.
Hope it helps.
VS
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