heavy chain on blot

[lerker]

Can anyone explain to me why you can get heavy chain coming up on your blot, when you haven't probed for it?
Thanks

[clokke76]

Hey, welcome to the club!

Are you using a secondary antibody that is specific for your species of primary antibody (if you are using mouse anti-protein X, are you using rabbit anti-mouse secondary)? If you are not, you may be getting non-specific binding of your secondary to your heavy chains.

From Roche Western Blot trouble shooting guide:


Heavy and light chains of primary antibodyvisible on blot membrane (Indirect detection procedures only)

Use direct detection with peroxidase-conjugated monoclonal antibody to visualize tagged proteins.

[admin]

Did you immunoprecipitate your protein first? this will greatly increase the amount of heavy chain that is present, and due to the fact that the primary antibody is bound to your protein, it is common that your antibody will ip with your protein of interest. When you blot, due to its abundancy on the gel, you may get non-specific signal similar to the way your ladder sometimes lights up and creates bands on your film.


There are means of removing it:
-cleave it with papain to decrease its size.

One paper did this at the heavy chain Fc was masking their protein of interest.

Doolittle et al., The Journal of Lipid Research, Vol. 39, 934-942, April 1998
" As a means of removing chicken heavy chain contamination at 66 kDa, the affinity-purified LPL antibody was subjected to papain cleavage to produce a truncated antigen binding fragment (Fab). Upon reduction, the truncated heavy chain of the resulting Fab was reduced from 66 kDa to about 25 kDa, well below the LPL band migrating at 57 kDa. "



If you didn't IP, then you might want to try another secondary or possibly primary antibody as they may be reacting to some Fc fragments of antibody.

See the next post for a method that works better but costs a bit more.

[admin]

Did you see something like this ie figure 2?

Figure 2: Stat1 was immunoprecipitated from 0.5 ml of 1x107 Jurkat cells/ml with 3 µg rabbit anti-human Stat1. Precipitate from 5x105 cells was subjected to electrophoresis, transferred to an Immobilon membrane, and immunoblotted with anti-Stat1 using Rabbit TrueBlot™ and conventional HRP-conjugated detection. Lane 1: Detection with Rabbit IgG TrueBlot™ - note the absence of the anti-Stat1 immunoprecipitating heavy and light chains. Lane 2: re-blot of lane 1 using the conventional HRP-conjugated detection anti-rabbit polyclonal antibody - note the presence of the Stat1 immunoprecipitating heavy and light chains confirming that although the immunoprecipitating heavy and light chains were present in the sample in lane 1, Rabbit IgG TrueBlot™ detected native antibody but not the denatured heavy and light chains.


if so you should use either papain or some kind of IP beads to get rid of the contaminating bands. True blot IP beads are an example.

We routinely use beads to pull down antibodies from IPs and then western blot only the proteins, the antibodies stay stuck on the beads.