5 replies to this topic

[cellgene]

Is it normal that sometimes I detect no light chain   ?

I know that my sample was reduced because I see the heavy chain about 50kDa.

thanks

[mdfenko]

heavy and light chain of what?

what are your separation conditions?

[cellgene]

mdfenko, on Mar 29 2010, 08:32 PM, said:

heavy and light chain of what?

what are your separation conditions?

opps sorry my question was obviously not clear

I perform an immunoprecipitation with protein A beads. Then I use reducing buffer and boil 5 minutes at 95 degrees. I load on SDS-PA gel.
I blot my gel and do westernblots.

First I developed the blot with the antibody that I also used for IP. So, I see the heavy chain of the immunoprecipitating antibody, but not the light chain. Is this normal?

When I repeated the experiment yesterday, I first developed the blot only using a secondary antibody just to see the IgGs, so I again only saw heavy chain.

It is important for me as one of my protein of interests is 22kDa so nearby lightchain.


Thanks.

[mdfenko]

your secondary antibody may be against only the heavy chain. have you tried running only the capture antibody for the blot or added a lane with only the antibody as a control?

what percentage acrylamide is your gel? your protein and the light chains may have comigrated.

what are the transfer conditions (membrane porosity, etc)? have you checked the membrane for protein (with ponceau s)? the small proteins may have blown through the membrane and not been captured.

do you have any images you can show us?

more information is necessary.

[cellgene]

mdfenko, on Mar 30 2010, 05:23 PM, said:

your secondary antibody may be against only the heavy chain. have you tried running only the capture antibody for the blot or added a lane with only the antibody as a control?

what percentage acrylamide is your gel? your protein and the light chains may have comigrated.

what are the transfer conditions (membrane porosity, etc)? have you checked the membrane for protein (with ponceau s)? the small proteins may have blown through the membrane and not been captured.

do you have any images you can show us?

more information is necessary.

one of my friends (who is not also very experienced) also suggested that secondary antibodies are mostly binding to Fc part of the primary antibody so it is usual not to see the light chain after the chains are separated. Do you agree?

I make a 12% acrylamide gel and transfer 2h semidry. I know that the proteins are still there because I see my protein on interest in the IP. The thing is, I have a negative control where I didn't overexpress the protein and there I do not see anything at the height of light chain (only thing I see is the heavy chain).
My protein is 22kDa.

Since I am not in the lab now I cannot send the picture but I will do it tomorrow.

Thanks again

[mdfenko]

cellgene, on Mar 30 2010, 05:24 PM, said:

one of my friends (who is not also very experienced) also suggested that secondary antibodies are mostly binding to Fc part of the primary antibody so it is usual not to see the light chain after the chains are separated. Do you agree?

yes.