Efficiency of the transfer in Western Blotting - How can you tell from the gel? (Jan/05/2006 )

My senior told me that after the transfer in Western blotting, the membrane shoudl be blocked to block unspecific binding, but the SDS gel stained in COmmassie to check the efficiency of the transfer? So how do youa ctually tell the efficiency when you stain, destain and dry the gel? How does the gel look when the efficiency of the transfer was poor and how does it look when it's good?

Also, can Commassie staining be always trustworthy? Sometimes it seems that it doesn't work good, like today I've stained my 3 gels from yesterday's transfer and it seems like they are stained good at the top and almost nothing in the middle. Does that mean the staining didn't work well or that the membrane actually got everything from the gel?

there are several ways to control that the transfer is OK.

1. you can color the gel with coomassie blue. If the transfer would be 100% you should see nothing. however, high molecular weight proteins are more difficult to transfer and a small amount stays in the gel. It seems your transfer was OK (especially if you don't see anymore proteins with the same weight than your protein of interest)

2. you can color transiently the blot with Ponceau red. You can buy it ready to use. sink the blot in ponceau red just after transfer, for 10 minutes. It becomes completely red. wash with water, proteins remain red, on a white membrane. check the transfer is OK. you can also check that there is no bubble on the transfer. you can continue to wash with water to wash completely the color. Some persons are washing and washing until color disappear completely, I start blocking even if there is still a litlle of pink on the membrane. after blocking it disappears.
This is the best method to check (this is my opinion)

3. you can use prestain molecular weight markers.
if you are careful, this method allows to check carefully without separating completely membrane from gel and continue the transfer if not OK.

I transfer with 0.8 mA / cm2 of membrane, for 1 hour and it's really fine.

all the best

laurence

I would like to add my questions:
1. Is dried and freezed membrane still good for immunoblotting?
2. Which electric parameter ie. voltage/current/power should be stabilized and on what value? (Bio-Rad manual says 10-15V for mini-gel but some other books say 0,8mA/cm^2 of gel sufrace...)

question1 : it's ok. I keep mine stripped and wrapped in saran on the bench

Before or after blocking?

If I do a transfer, I blot it whithin 3 days, and I keep it in blocking buffer, in the cold room.

However, I already re-blotted blots that I kept dried, or blots that I kept in the saran in -80°C(after ECL detection) (it was for phosphorylation purpose) after several weeks. It works.

about transfer, it is said that you should transfer with 0.8 mA per square centimeter, one hour.

I usually do 150 mA one hour for one blot, 1h30 for 2 blots, on SDS-PAGE, and I don't soak the gel in transfer buffer (tris glycine plus 10% methanol) before transfer. SDS inside the gel helps for the transfer.

Laurence

I usually do 150 mA one hour for one blot, 1h30 for 2 blots, on SDS-PAGE, and I don't soak the gel in transfer buffer (tris glycine plus 10% methanol) before transfer. SDS inside the gel helps for the transfer.

Laurence
[/quote]
i just began to do western blot a few weeks ago,we are studying Dnmt3a,i tranfer it at 250mA,2h
i want to know if the parameter is proper?And i usually soak the gel in Tris glycine transfering buffer for a few minites before transfer,does it have any difference?
Thank u!

[quote name='changjian2' date='Mar 7 2006, 07:27 PM' post='43422']
I usually do 150 mA one hour for one blot, 1h30 for 2 blots, on SDS-PAGE, and I don't soak the gel in transfer buffer (tris glycine plus 10% methanol) before transfer. SDS inside the gel helps for the transfer.

Laurence
[/quote]
i just began to do western blot a few weeks ago,we are studying Dnmt3a,i tranfer it at 250mA,2h
i want to know if the parameter is proper?And i usually soak the gel in Tris glycine transfering buffer for a few minites before transfer,does it have any difference?
Thank u!
[/quote]


When I started my PhD, I was doing the same. 250 mA and I was soaking few minutes, (meanwhile I was preparing the sandwich for the transfer). In fact I've learned it's useless.( it will not damage your transfer.)

Thank u for your answer,Mr.Missle