38kda protein appearing as smear on western - (Feb/01/2006 )
Hello,
i have a very strange problem! im doing western blotting for some time now but previously i
looked for proteins with a big
molecular weight.
Now i want to check a small protein eif2alpha ~ 38 kD, and the trouble is that i am seeing a smear
instead of distinct band.
i am running 4-20% precast gels and tranferring using PVDF paper for 1 hour 30 mins at
140milliamp constant current.I was using this method previously and was working with the higher
mol wt proteins.Transfer buffer is Towbins with methanol.
the strange thing is that the marker is there even the smaller band at 10kda.
I made all new running buffers,sample buffer,ripa buffer for protein isolation etc but the problem seems to be recurring,i am so frustrated,please help me.I am doing this for more than 2 months.
Changed protein isolation technique,used better primary aby dilutuions.,but nothing seems to work. i use protease inhibitor cocktail from roche to prevent protein degradation.I block with 5% milk in PBS and 0.1%tween-20 soln.
if anyone doing westerns for small proteins can you please give me some hints?
i have a very strange problem! im doing western blotting for some time now but previously i
looked for proteins with a big
molecular weight.
Now i want to check a small protein eif2alpha ~ 38 kD, and the trouble is that i am seeing a smear
instead of distinct band.
i am running 4-20% precast gels and tranferring using PVDF paper for 1 hour 30 mins at
140milliamp constant current.I was using this method previously and was working with the higher
mol wt proteins.Transfer buffer is Towbins with methanol.
the strange thing is that the marker is there even the smaller band at 10kda.
I made all new running buffers,sample buffer,ripa buffer for protein isolation etc but the problem seems to be recurring,i am so frustrated,please help me.I am doing this for more than 2 months.
Changed protein isolation technique,used better primary aby dilutuions.,but nothing seems to work. i use protease inhibitor cocktail from roche to prevent protein degradation.I block with 5% milk in PBS and 0.1%tween-20 soln.
if anyone doing westerns for small proteins can you please give me some hints?
I recently discovered that loading too much protein may cause some bad smearing, especially when your antibody is not so great (all the eIF2alpha antibodies that I know of are not very good). So lower the protein you load in each lane, I've tried to decrease from30ug to 5ug, which worked really well. Also, when you do blocking, primary and secondary incubation, do not use milk, because milk will interfere with phosphorylation detection (i assume you're trying to find phospho-eIF2a), use BSA instead.
How much SDS is in your extraction buffer and sample buffer? I have found that the less SDS I have the more things smear. I have found that a 1.0% SDS in my exraction buffer works great and I don't need to put any in my sample buffer. If my SDS is lower, I need to use a sample buffer with more SDS in it.
I am also seeing smears for proteins less than 30 KDa on both my SDS-PAGE and Western membrane.
how does your gel look like?
I've been having this problem for 1 year now. I might upload some photos next time.
my protein is 25KDa, I run 12.5% gel. 100V for 1:30 hrs. then I use PVDF membrane and run 15V 60mA for 1 hr. I can detect the protein. even actin protein is there, but still my protein appears like a thick smeared band.
Try staining the gel with Coomassie or stain the membrane with Ponceau S and see what the bands look like in the lower region... if they are smeared then it is a problem with running the gel and/or the transfer. If they are not smeared, then maybe it is due to post-translational modification (ubiquitylation, sumolyation, phosphorylation... etc.).
i have a very strange problem! im doing western blotting for some time now but previously i
looked for proteins with a big
molecular weight.
Now i want to check a small protein eif2alpha ~ 38 kD, and the trouble is that i am seeing a smear
instead of distinct band.
i am running 4-20% precast gels and tranferring using PVDF paper for 1 hour 30 mins at
140milliamp constant current.I was using this method previously and was working with the higher
mol wt proteins.Transfer buffer is Towbins with methanol.
the strange thing is that the marker is there even the smaller band at 10kda.
I made all new running buffers,sample buffer,ripa buffer for protein isolation etc but the problem seems to be recurring,i am so frustrated,please help me.I am doing this for more than 2 months.
Changed protein isolation technique,used better primary aby dilutuions.,but nothing seems to work. i use protease inhibitor cocktail from roche to prevent protein degradation.I block with 5% milk in PBS and 0.1%tween-20 soln.
if anyone doing westerns for small proteins can you please give me some hints?
I am not familiar with your protein but smearing may result from posttranslational modifications, in your case multiple phosphorlyations are possible; check if your protein is separated in 2D over a broad range of pI