western blot explenation - (Jan/29/2013 )

I have a question on a western blot analysis from a paper I am reading.

I can not understand where the "75" protein comes from in the picture.
The 50 kDa protein is the protein they looked for, so ok, I understand it pops up , but where does the 75kDa protein comes from?
They incubated it with anti MBP (=50kDa protein) sera, so where does the 75kDa comes from?

A second question is the following, in their explenation they mention the following (see red bold text).
What does that mean? they incubated the MBP antisera with P. putida (neg controle) lysate? Why would they do this? WHats the point of incubating the antisera with lysate from a bacterium that does not contain the MBP?



Expression of MBP-EC20. Western blot analysis was used to probe the expression
of MBP-EC20. Samples were centrifuged at maximum for 5 min, and
the supernatant was discarded. The pellets were stored in a freezer set to _20°C
until further processing. Samples were concentrated to an OD600 of 20 and
boiled at 95°C for 10 min. The cell lysate was loaded onto 12% (wt/vol) polyacrylamide
gel (22), and the proteins were separated by sodium dodecyl sulfatepolyacrylamide
gel electrophoresis. Proteins were then transferred to a nitrocellulose
membrane and incubated with rabbit MBP antisera (New England
BioLabs, Beverly, MA) preincubated with P. putida wild-type cell lysate.
Western blot analysis was performed using an Immun-Blot GAR-AP kit
(Bio-Rad, Hercules, CA).

The 75 kDa bands are most likely cross-reactive bands, i.e. non-specific binding of the antibody. Pre-incubation with a negative control is to help remove cross-reactive bits in the antiserum.

When you make an antibody, unless you are making a monoclonal antibody from a hybridoma, then you also get all the other antibodies that are floating around in the animal at the same time, so you actually get a lot of cross-reactivity unless you can purify the antibody or find appropriate blocking and dilutions that minimize these bands while still giving strong signal from the specific band.

bob1 on Tue Jan 29 19:29:36 2013 said:

So you are saying they did the pre-incubation to remove all the antibodies (= should be the antibodes against the MBP) that would react with unspecific proteins (would react with other proteins then the MBP)?
Right?

I can see this, but they did purify the antibody before (at the commpany) too? To make sure that you dont have all the other antibodies in the anti seria MBP , right?

Correct, the pre-incubation was to help remove antibodies that will bind to proteins other than MBP that are present in the lysate. The anti-serum in question has not been further purified to produce purified antibodies against MBP, if it is the one I found at NEB (cat number E8030S), it would say "Affinity purified" if it were.

An anti-serum is different to an antibody. Anti-sera are where the blood has been collected from the immunized animal and the red blood cells removed, but nothing else. An antibody is a small component of the anti-serum that binds specifically to a particular sequence. In all anti-sera, there are many many many antibodies against all sorts of things from food the animal is eating, bacteria, viruses, fungi, dust... you name it, it's there. A typical antibody recognition sequence is less than 15 amino-acids in length, often as few as 4 aa's, which as you can imagine, is not very specific. This is the reason that either further purification is needed, or some sort of pre-incubation to help remove antibodies that bind to proteins other than the one you are looking for.

bob1 on Tue Jan 29 21:05:24 2013 said:

Ok I see.

Because it was an anti sera (and not a specific antibody) they used the pre incubation to get rid of all the parts (all the other antibodies) in the sera that would bind with the "normal" DNA coded proteins of the bacterium.
So that only the antibody that would bind the MBP was left in the end. (well theoretically speaking, as there is still some aspecific binding)

Yes. Though as you can see from the blot - there are still non-specific bands, so either there was too much protein in those bands to be removed, or those proteins have similar site(s) to the MBP antibody recognition sequence (called an epitope).

bob1 on Tue Jan 29 21:25:10 2013 said:

Ok, thanks.

Altough, I have to admit I find it weird that the aspecific band would be 1 band at the same height.
I would think you would have aspecific binding pretty much everywhere since you do load all the proteins on that nitrocellulose membrane. So it is strange that only at a certain size you have this aspecific binding.

I understand what you said about the "having a similar site to the recognition sequence, however
I am not sure what you mean by this:

I do not understand it.
To my knowledge (as I also said above in this post) you blot all the proteins on the nictrocellulose membrane , so I do not understand what you mean by too much protein in those bands to be removed..
You only see bands because the anti sera was able to bind at those specific places with proteins, right? So why would it matter to have a lot (too much) protein at the nitrocellulose membrane?

Sorry, got that bit wrong: What I was meaning to say was too much antibody to be completely removed during the pre-incubation.

bob1 on Tue Jan 29 21:55:49 2013 said:

Ok.
I got it, thanks.