Dear Pilar,

Unfortunately, your question is not sufficiently specific to solicit productive answers. What I can do is to try deriving more specific question for you by creating a process that may lead to the resolution of your problem.

1) First we have to establish what are your binding partners. Do both constructs BSA as well as TLL represent full sequence proteins or some truncated products (for instance the binding domain of TLL).

2) What are your crosslinking agents?

3) How did you characterize the aggregates, (size, concentration, how much of the remaining proteins in solution etc)?

Possible experiments that might help you to attack your problem:

1) Quantifying the percentage contribution of different proteins to your aggregate. Collect aggregate eliminate crosslinks and quantify the protein by SDS gel elctrophoresis.

2) Build (or obtain from the PDB or some model building server) models for both proteins and look for the location and density (how many) of side chains that can accommodate crosslinking groups.

3) Change of crosslinking agents (crosslinking different groups). By counting all possible crosslinking groups on the surface you can get an estimate of probablilistic chances for crosslinking and, therefore get a handle on the stoichiometry of your aggregate. For possible crosslinking agents please examine:

http://www.piercenet.com/browse.cfm?fldID=0203

4) Instead of radio-labeling your proteins use a fluorescent tag for your protein.

5) Estimate the size and shape of your aggregate by small angle light or X-ray scattering methods.

6) Change (scan) crosslinking conditions. Titrate both protein components as well as the crosslinking agent.

It seems to me that the BSA is a much larger protein and you want to use it as a carrier for TLL binding unit. If you use a crosslinking agent that has a single binding group on the TLL and many on the BSA you can probably create a stoichiometry you desire. Good Luck.

Dear Pilar,

Unfortunately, your question is not sufficiently specific to solicit productive answers. What I can do is to try deriving more specific question for you by creating a process that may lead to the resolution of your problem.

1) First we have to establish what are your binding partners. Do both constructs BSA as well as TLL represent full sequence proteins or some truncated products (for instance the binding domain of TLL).

2) What are your crosslinking agents?

3) How did you characterize the aggregates, (size, concentration, how much of the remaining proteins in solution etc)?

Possible experiments that might help you to attack your problem:

1) Quantifying the percentage contribution of different proteins to your aggregate. Collect aggregate eliminate crosslinks and quantify the protein by SDS gel elctrophoresis.

2) Build (or obtain from the PDB or some model building server) models for both proteins and look for the location and density (how many) of side chains that can accommodate crosslinking groups.

3) Change of crosslinking agents (crosslinking different groups). By counting all possible crosslinking groups on the surface you can get an estimate of probablilistic chances for crosslinking and, therefore get a handle on the stoichiometry of your aggregate. For possible crosslinking agents please examine:

http://www.piercenet.com/browse.cfm?fldID=0203

4) Instead of radio-labeling your proteins use a fluorescent tag for your protein.

5) Estimate the size and shape of your aggregate by small angle light or X-ray scattering methods.

6) Change (scan) crosslinking conditions. Titrate both protein components as well as the crosslinking agent.

It seems to me that the BSA is a much larger protein and you want to use it as a carrier for TLL binding unit. If you use a crosslinking agent that has a single binding group on the TLL and many on the BSA you can probably create a stoichiometry you desire. Good Luck.

Thank you so much for your answer Proff Stec.

Indeed, I used BSA as carrier for TLL using glutaraldehyde as crosslinking agent. BSA has the highest Lys residues,then we propose BSA is the core of aggregate.

Now, is probably that I can characterize the aggregates with SDS gel electrophoresis as your suggestion!!!

A characteristic of serum albumin that you can utilize for labeling and crosslinking is that, unlike most proteins, it has one unpaired cysteine.