Insufficient transfer in Western blotting - (Mar/02/2007 )
HI there, I've got big problems here. Ihave been running Western for 4 months, and this is the third time I have these) problems:
1) I transfer two gels/run into two membranes, and it seems to me that the transfer is very good in one gel (closer to the positive node) and is insufficient in the remaining (in negative side). My transfer conditions: 25V for 75mins and I am looking for a protein of 27-29kDa.
2) In the gel with sufficient transfer, the bands on the blot look very nice. Nevertheless, with insufficient transfer, the other blot has diffuse bands, grainy looking smear along two sides of the bands. I confirmed the transfer with Commassive Blue stain for the gels and the gels got smears down the low molecular weight regions (which is the region of my protein to be).
3) Big bands of around 60kDa (which I guess could be albumin). how to get rid of this.
4) Alot non-specific bands up in high molecular regions (>60kDa).
My Western conditions for cell lysates
1) Load 20ug of protein/well. Run Positive control. Electrophoresis: 200V, 1h, 160mA.
2) Transfer 25V, 75mins. Nitrocellulose membrane. The membrane is soaked in trhe trasfer buffer at least 15-30 mins before transfer. Membrane air dries abt 10mins
3) 2hrs blocking 5% skim milk in TBST) 1X5mins wask TBST
4) Overnight incubation in primary antibody (1:1000 in 1% skim milk). 10-10-5mins wask in TBST (10mls/wash)
7) 1h incubation in Secondary (1:2000 in 5% skim milk), 10-10-5mins wash in TBST.
5) 1min ECL exposure
Need urgent help as i dont have much time left for my honours.
are you running the transfers in the same tank?
do you wash the gels in transfer buffer before you blot to get rid of the SDS?
it sounds like maybe your transfer apparatus is cranking out; perhaps borrow or buy another and see if it helps?
it also seems as though you have some problems with specificity/background. graininess aside, it sounds like you're getting way too many bands.
I would wash more after step 3. one wash probably isn't enough, even if it's for 5 min
what are the characteristics of your antibody? is it polyclonal?
also, I would reduce the time for the secondary; 60 min is a very long incubation time. I would do 15-30
good luck
do you wash the gels in transfer buffer before you blot to get rid of the SDS?
Yeh, I used the same tank for electrophoresis and transfer.
And I wet the gels with transfer buffer (2-3mLs), but no a real wash.
it also seems as though you have some problems with specificity/background. graininess aside, it sounds like you're getting way too many bands.
I would wash more after step 3. one wash probably isn't enough, even if it's for 5 min
what are the characteristics of your antibody? is it polyclonal?
also, I would reduce the time for the secondary; 60 min is a very long incubation time. I would do 15-30
good luck
My antibodies are all polyclonal, explaining why I have quite a lots of non-specific bands. But I run positive ctrl in evey run, so it should be ok.
I uploaded a pix of my run so you guys can have a better idea of what is going on. Blot 1.1 (front gel) and blot 1.2 (back gel).
it also seems as though you have some problems with specificity/background. graininess aside, it sounds like you're getting way too many bands.
I would wash more after step 3. one wash probably isn't enough, even if it's for 5 min
what are the characteristics of your antibody? is it polyclonal?
also, I would reduce the time for the secondary; 60 min is a very long incubation time. I would do 15-30
good luck
My antibodies are all polyclonal, explaining why I have quite a lots of non-specific bands. But I run positive ctrl in evey run, so it should be ok.
I uploaded a pix of my run so you guys can have a better idea of what is going on. Blot 1.1 (front gel) and blot 1.2 (back gel).
Hi
It looks like the side close to the electrod have got a good buffer circulation which will enable more efficient transfer ( sharp bands, and more protein). did you use the cooling coils ( or ice cooling system) it will help.
Also, are you running mini gels, 200volts to run the gel is too much, this might give the diffused bands, try and run at 100volts you get better separation that way too.
You can't get rid of albumin, unless if you purify your protein by immunoprecipitation or something.
try to minimize your proten load becaue you are getting alot of non-specific binding too.
block in 5% skim milk in 1X TBS without tween. one hour should be fine. also don't wash your blocking, just rinse the membrane with 1X TBS quick rinse, then add your primary. if your primary react with other stuff, block for shorter time, 2-3 hours. or you can dilute more (i.e.1:2000). Are you using anti Rabbit HRP, I use that at (1:15000 times dilution). I get very nice clean blots. good luck
It looks like the side close to the electrod have got a good buffer circulation which will enable more efficient transfer ( sharp bands, and more protein). did you use the cooling coils ( or ice cooling system) it will help.
Also, are you running mini gels, 200volts to run the gel is too much, this might give the diffused bands, try and run at 100volts you get better separation that way too.
You can't get rid of albumin, unless if you purify your protein by immunoprecipitation or something.
try to minimize your proten load becaue you are getting alot of non-specific binding too.
block in 5% skim milk in 1X TBS without tween. one hour should be fine. also don't wash your blocking, just rinse the membrane with 1X TBS quick rinse, then add your primary. if your primary react with other stuff, block for shorter time, 2-3 hours. or you can dilute more (i.e.1:2000). Are you using anti Rabbit HRP, I use that at (1:15000 times dilution). I get very nice clean blots. good luck
I tried every thing, and also had someone (expert in Western) to watch over me, but it didnt help much. I didnt do any wrong.
I guess the modules are old, and cracked. I will do another run in the newer system to see if there is any improments.
1) I transfer two gels/run into two membranes, and it seems to me that the transfer is very good in one gel (closer to the positive node) and is insufficient in the remaining (in negative side). My transfer conditions: 25V for 75mins and I am looking for a protein of 27-29kDa.
2) In the gel with sufficient transfer, the bands on the blot look very nice. Nevertheless, with insufficient transfer, the other blot has diffuse bands, grainy looking smear along two sides of the bands. I confirmed the transfer with Commassive Blue stain for the gels and the gels got smears down the low molecular weight regions (which is the region of my protein to be).
I ussally use the transfer buffer containing:
Glycine = 39mM
Tris-base = 148mM
SDS=0.037%
Methanol = 20 %
Transfer at 100V for 1h with ice cooling unit inside and "ice + water" outside the chamber. It worked well before.
Recently I didn't use ice pocket (because it's broken), I've just put the chamber inside the box containing Ice + water (but I think the buffer is cool enough). However, unluckily, I got the same problem with you. Just I don't remember exactly in which side the gel got this problem.
In the bad transfer membrane, seem half of lanes gather at one point, the other half have interrupted transfer because of bubbles even though I remove air bubbles before transfer.
My senior recommend that use the Towbin's transfer buffer, and it buffer need to be kept at 4oC before using. During transfer, using not only ice cooling unit but also the magnet bar to make the steady condition.
I hope you (and me also
) could overcome this problem ^^