Hi

I used western blot for measuring the activity and expression of intracellular signaling pathways molecules and for this purpose I used 100V and 0.35A for 1 hour for transfer of protein size

43 KDa.

In general, detection of proteins by Western blotting is emperical (and less science). Although, there are number of protocols, each of them has numerous variables. Therefore, one needs to optimize- sample preparation, gel running conditions, transfer conditions, and immuno-detection conditions. The link below provides the relevant information for an optimized protocol.

Link: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6376.pdf

Hello,

Could you recheck your sandwich preparation for transferring. I also used same voltage and time and observed the desired protein band. You also can give a look on your cell lysis buffer composition if you are using less protease inhibitors. This might cause denaturation of the protein and you will not get the exact band.

using a precast TGX gel for your SDS-PAGE  may give you a better result, also try running your gel between 120Volt to 150Volts. Goodluck

Hi to run gel I use Constant Voltage 100V, to transfer I used programs of TURBO TRANSBLOT Biorad 

Hi Iwan!

Other important parameters in gel transfer are the percentage of acrylamide, the composition of transfer buffer, the type of apparatus.

Normally, I use 70V for 1h 20' and I can see proper transfer

of proteins from 15KDa to 120 KDa. I add methanol in transfer buffer and I use ice to cool the apparatus during transfer. Please, can you describe your working procedure for a better trouble shooting?

hi silvia,

i hope these further information will be sufficient, if not do let me know and i will be eager to share with you more. i also use methanol in my western blot transfer buffer. I recently tweaked the protocol for transfer by using 80V for 2 hours for protein sizes ranging from 50kd to 110kd. i see some faint bands for 110kd but for  a 50kd protein i did not see any bands.

actually it may or may not be due to the transfer voltage used as my actin expression is also as low as the faint bands seen for the 110kd proteins by antibody detection. so i have a lot of work to do.

however i will appreciate it if anyone can provide a 'safe voltage'' for transfer of proteins size 43kd-110kd.

80V for 1 hour

Hi Iwan

I will recommend you to use 70 V for 70 minute for protein band lower than 110 KDa. For methanol concentration, you have to use 10% methanol in transfer buffer. I think 80V for 2hr is more to analyze the lower molecular weight protein. I will also suggest you before blocking, you should stained your gel with cb and membrane with ponceau to see whether transfer is ok or not.  if you see the good band in membrane then go ahead. Good Luck

Hi Iwan

In your case the possibilities could be

1- the transfer was not working ( which means the protein was not transferred to the nitrocellulose paper) I recommend trying longer transfer time ( 1 hour and 45 minutes, 300 AM and put some ice in the tank of transfer buffer)

I wish this would help

best 

1. Inlcude pre-stainined protein marker ladder in gel run, which will tell whether gel run is OK or not.

2. I guess you are doing wet transfer, then use cold buffer and a cooling pack to avoid over-heating of the system during the transfer (1.5hr at 100V is fine for Towbin-tranfer buffer).

4. After transer, stain the blot with ponceau (which stain the protein bands reversibly, which disappear by incubating the stained blot in TBS) to ensure proper transfer (pre-stained markers also confirm this).