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Multiple bands on Western blot for an expressed protein - (Sep/01/2006 )

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Hi!
I'm asking for help. I'm trying to check my cloning and transfection efficiency by running Western Blot. Today I have seen the results:(. Leaving behind the cloning, I sow multiple but real multiple bands on my film. I washed the membrane 5x5min, as blocking buffer used 5% milk, 4% BSA, 5% Tween-20, but still. I can see bands of smaller sizes than my protein, and very dark line indicating loading. Don't know what to do about that:( Please if you have comments....

-malena.a-

could they be degradation fragments, if they are all smaller?

could your antibody be too non-specific? is it polyclonal?

-aimikins-

QUOTE (malena.a @ Sep 1 2006, 11:23 AM)
sad.gif [font=Courier New][size=3]
Hi!
I'm asking for help. I'm trying to check my cloning and transfection efficiency by running Western Blot. Today I have seen the results:(. Leaving behind the cloning, I sow multiple but real multiple bands on my film. I washed the membrane 5x5min, as blocking buffer used 5% milk, 4% BSA, 5% Tween-20, but still. I can see bands of smaller sizes than my protein, and very dark line indicating loading. Don't know what to do about that:( Please if you have comments....


Hi,

It may because:

1. over-loading of sample, try smaller amount.
2. 2nd antibody, try to decrease 2nd antibody and some unwanted bands will disappear.

I have multiple bands in the past, but most of them would disappear after I decreased the sample and 2nd antibody.

-rabbit-

unspecific bands have become part of my life
look for the right one and move on with your life
you should se e it comparing transfected vs untransfected samples considering the molecular size fo your protein

-tertu-

Hi!
My antibody is polyclonal rabbit, thanks I will try to do my best.

-malena.a-

improving Westerns is always a good idea but are your additional bands really unspecific?
Degradation was discussed; is your vector pure? do use various similar vectors? are contaminations excluded? recombinations in cells of your successfully transfected vector may happen resulting in different sized bands; try a monoclonal Ab; do your protein carrys a tag? and is an Ab available?

-The Bearer-

run negative control and see the difference, if there bands are only in your induced sample than i think it is degradation products of your protein. but polyclonal antibodies are PIA.

-Kathy-

hello,

a few questions: what organism are you using for your experiments? also, what vector are you using for your expression?

if this is e. coli, it's not uncommon for you to recover multiple truncated forms of your expressed protein. sometimes, re-transforming w/ fresh vector will help. also, if your tag is located on the N-terminus, you will see all of these products. to get around this, you can use an antibody directed to the C-terminus (and a C-terminal fusion tag if you can...that way, you'd only be purifying the full-length fusion proteins).

if this is protein is expressed in something other than e. coli, it might be getting proteolytically degraded, either in vivo, or in vitro, depending on the presence/absence of your proteinase inhibitors in your extraction buffer.

good luck

QUOTE (malena.a @ Sep 1 2006, 02:23 PM)
sad.gif [font=Courier New][size=3]
Hi!
I'm asking for help. I'm trying to check my cloning and transfection efficiency by running Western Blot. Today I have seen the results:(. Leaving behind the cloning, I sow multiple but real multiple bands on my film. I washed the membrane 5x5min, as blocking buffer used 5% milk, 4% BSA, 5% Tween-20, but still. I can see bands of smaller sizes than my protein, and very dark line indicating loading. Don't know what to do about that:( Please if you have comments....

-johanski-