We call this of inespecific binding. Some times these non-specifics bands arise more stronger than the specific band. Unfortunately.

Protein degradation as well as some unspecific binding are the two possibilities, I can think of. Try using a monoclonal antibody or more stringent steps in washing and dilutions to get a better band.

may be try higher dilution of secondary antibody to get off that non-specific bands

1. Usually in my case it bcz of inappropriate washing after the incubation of secondary antibody.

2. Dilute the solution A and B (that also work sometime) in this case you will require more time about 5-10 sec when reading your blot.

Do you use 5% BSA or milk as antibody diluent for both ab? It helps in reducing the background. In addition, to proper washing, the secondary ab dilution and incubation time might also affect in your case.

@ Vinoth M. - You really use 5% BSA or Milk as diluent ?????

I am perfectly in agreement with Miguel about all he said.

In your explanation you did not talk about any kind of positive/negative controls which can help you to understand if it really an unspecific band (which happends often if you use polyclonal antibodies, as already said) or it can be related to your protein.

If you follow the suggestion from Miguel and you still have doubs, you can cut the band, digest and see it on MS (of course, whether your lab may do it).

Anyway, the purity of a protein is difficult to assest by WB. Side-exclusion chromatography coupled with absorbance schift measurements may be one of the right choice. By the way, the method which you use to purify your protein can be useful for us to help you.

Cheers

hey,

first of all thanks for the fast response...

I`ve already "blocked" with milk before treated the WB with antibody, and I guess that it is a "fresh" problem. Anyway I`ve started the WB again and let you konw if the "mythic" fragments disappear ;)

@Miguel: Fish-Proteins? That sounds interesting, but you are right :)

@Stella: the purification was with a special "gel-matrix" (anion exchange resin and phosphocelluose resin)

cheers...and thx for the help

Martin

great explanation Muguel..

...only one more thing

in my esperience

you can also overload on SDS-PAGE your protein (more then 10 ug/well) adding fresh beta-Mercaptoethanol (1ul/25 ul sample). The coomassie stain gel should show only one big band of your protein. If it contains contaminants you will see them and you can estimate the purity (95%....99%)

Thanks Paola,

that "overload method" is new to me. My last WB was (luckily) free of the upper light fragments, so I think it was an unspecific antibody-binding-phenomena.

you are welcome Martin

in wb you detect only fragment related to your protein, often these are even undetectable in coomassie.

What I used to do:

when I was sure to see, after staining the gel , only one band I performed a western blot to be sure of the i protein dentity and to detect degradation.

I agreed Miguel

the "very light fragments above the main fragment" will disappear If you add fresh b-Me to your sample just before loading. Anyway your data suggests that your protein forms multimers by Cystein bridges, check it by a WB of a non-reducing SDS-PAGE (don't run reducing and non-redicing samples in the same gel)

Ciao!

H Paola,

Just out of curiosity...What will happen if reducing and non-reducing samples are run in the same gel?

Hi Arpita

science is curiosity

b-Me diffuse to neighboring samples during the run in SDS-PAGE, during loading,

many "old researchers" prefer to run non-reducing and reducing gels in different electropheretic chambres,

that was taught me

try to believe.....

Hi Paola,

hm I add ß-Me to the samples before I cook it but maybe it wasn`t fresh, anyway.

So it is better to add it instantly before loading?

In my last WB the higher fragment wasn`t there. It is also amazing (but also logical) how much the blot depends on the amount of the sample you load on, I dilute the sample one step further and the fragments (protease activity) under the main are more or less lost.

"science is curiosity" yes it is ;)