band spread in western - (Mar/07/2005 )

Hi,
I am doing western on a potassium channel subunit. I can detect the band. However, the band kind of spreads out compared to the marker next to it. Actually, the band width is twice the width of lane. Anyone here knows why? Thank you very much.

Hi,

I have seen this several times with my protein. It happens when the salt concentration is high into the sample that you load on the gel. I suggest you to dilute as much as you can your sample or make a dialysis of your sample, it may help you.

Hi, Dan:
Thank you for your reply. I checked my recipe and found the NaCl concentration is about 140mM. Do you think it is too high? Also, could it be because that the I added 30ul sample, but only 10ul marker into each well? So that only the sample spreads, but not the marker.
Thanks.

Hi hjdeal,

140mM of NaCl is certainly too high. You should tried, but I'm sure increasing the quantity of your loading buffer won't help you. Try to make a dialysis of your sample or a protein precipitation.

The marker doesn't make any difference....

gook luck!

Hi, Dan:
Thank you very much for your advice. I will try to dialysize my sample and lower the salt concentration. By the way, do you have any experience with using Amicon Centricon columns, which are claimed to be able to concentrate and desalt solutions?
I am appreciated to your help.

Hjdeal

hi hjdeal,

I have some experience with Amicon Columns, but I do not use these column to dialyse proteins samples, but I can tell you to use amicon with MWCO at least 2 times mores than your MW of your protein, because I have used Amicon columns with 30 kDa MWCO and have, after separation, I recovered about 30 to 40 % of BSA into the filtrate. hope that your protein haven't a low MW!

ciao!

I use Amicon centricon units for concentration and diafiltration routinely. works very well, but as dan_ mentioned, you should use low MWCO columns, since the MWCO is only an approximation. Using columns with a MWCO of at least half the MW of my protein gave me good results, in fact I use 5kDa cutoff columns most of the time for proteins ranging from 20 to 130 kDa.

The other thing you could try to do is to put your standard into the same buffer as your samples. lanes spreading or narrowing is caused by different salt concentrations in neighbouring lanes. If everything's in the same buffer, all lanes run the same - i.e. in my hands at least!

mike

Thank you very much, dan and mike.
I will give a try. Actually, the proteins I am looking at are 38kDa and 160kDa respectively. I ordered the column with MWCO at 10kDa. Hopefully it will work.
I will post my experience after I try the column.

Could your protien be glycosylated? If so the wide band would represent that, try deglycosinase such as pgnase (NEB) and see if it shifts down with treament.

Hi
the band spread that much could be from too much protein load. try and dilute your sample a bit more and see what comes up.
to clean up your sample you could also use acetone precipitation.
its much easier, then solubilize your protein in sample buffer