Hello Divya,

The heaviest protein that I have analyzed by WB is a 210 kDa protein. But I detected without problems, my protocol was:

Wet transfer with transfer buffer: Tris  25 mM, Glycine 192 mM, Methanol 20%.

The blotting could be perform in two different ways:

90-100 V, 2h 30 min, 4ºC  

30 V, Overnight, 4ºC

Personally I preffer the second one, because you don't heat up the transfer buffer, and in general you have a complete transference to the membrane. Do not use SDS. This protocol is optimized for a NC membranes. Do not forgot to check if the transference worked, i.e staining the membrane with Ponceau red (it can be removed later with TBS-Tween). And if your protein is a membrane protein, sometimes microwaving the membrane rinsed in PBS is usefull to increase the detection (check the bibliography).

I hope this will help you                     

Hello DIvya, 

Also, check the pI of your protein. If the pI is near the pH of your transfer buffer (8.3), you may need to try another buffer (such as CAPS/MeOH pH 11)

Presence of SDS, Methanol, Voltage/power and % of gel used effects the transfer of the protein to membrane.  Please try to use lesser % gel. For more than 200kD less than =

Use 0.05% tween 20 in your transfer buffer. It has been used to successfully transfer nearly 300 kda proteins in our lab.

i got good result for m-TOR which has molecular weight  of 289. we didn't used SDS in transfer buffer but just used in running buffer. generally  we do transfer by adding 200ml methanol per one liter of trans fer buffer. (for one liter we add 14gr glycine+ 3gr tris base+ add double or triple distilled water to get the final volume of 800ml + 200ml methanol). i personally used 10% gel .if you want to use you can use 8% also. we did transfer at 35V and the temperature is 4ºC over night. we used PVDF membrane. after transfer you do the next procedure  or you can also check with ponceau.  doing transfer at higher voltages and less time may cause improper tranfer and heating.

not only transfer every step(running, transfer, blocking , 1º -Ab, 2º-Ab treatment washings, final substrate addition) is important and as of i know its better to use CST-Abs please check the literature and google images for getting information on mlot pattern.

loading dye or lamelli buffer proper preparation also important.

running buffer= 14gr glycine+3gr tris base+water

transfer buffer=14gr glycine+ 3gr tris base+ 200ml methanol+water

try with PVDF membrane and soak the membrane in methanol for ~5minutes.

PBST= 1X PBS one liter+ 250 micro liter tween.

blocking= 5gr milk powder + PBST to make final volume 100ml.

5% milk is better than BSA  for blocking as BSA is giving background. prepare Ab solutions and blocking solutions in PBST. 

i think it works. all the best. 

if you have any doubts please contact [email protected]

I would add just one point to the protocols Sudhakar just described. After the milk powder is completely dissolved in either PBST or TBST, check the pH. You may find that the milk powder has reduced the pH by as much as 0.5 units. (At least, Carnation Skimmed Milk Powder has that effect on our TBST).

Thank you everyone. I will check all these methods.

Hi Divya

Curious: how do you know your protein is present in your sample in the first place?Transferring a 260 kDa is pretty simple (in theory at least; used to work on a 450 kDa without major issues) and should not be a major problem.  Just make it slow and steady (overnight wet transfer or low current/longer  "dry" transfer); it will help to use lower percentage gels, for resolution purposes mostly.

But first, you should first make sure that 1- your protein is expressed sufficiently in the model system by some other method (you could use microscopy with a siRNA control, look at public mass spec databases etc...) and 2- that the bulk of your proteins around the 250 kDa are transferred properly with your favorite membrane compatible protein stain.  

Make sure you include a positive control of some sort (sample known to contain the protein of interest, recombinant fragment etc..) if you keep on having problems detecting it (your antibody may be dead or of poor quality).

Good luck