4 replies to this topic

[henry-fan]

hi ,everybody.
currently ,I start to do a poject about two protein interaction ,first I want do a coIP, the protein is attached to different tag. such protein X with myc and protein Y with FLAG.
     since I didn't do this kind experiment , I am not very certain of  how to set control in different period : cell transfection , IP and western blot.
    any comments and suggestions are welcome
    thx

[cellcounter]

henry-fan, on Jul 31 2009, 11:55 PM, said:

hi ,everybody.
currently ,I start to do a poject about two protein interaction ,first I want do a coIP, the protein is attached to different tag. such protein X with myc and protein Y with FLAG.
     since I didn't do this kind experiment , I am not very certain of  how to set control in different period : cell transfection , IP and western blot.
    any comments and suggestions are welcome
    thx

1. Transfect cells with two constructs (test), cons X and cons Y only (negative controls), and untransfected cells (negative control).

2. Lyse, divide each lysate in two aliquots. IP each aliquot with either myc or flag antibody.

3. Run the IPed protein on SDS-PAGE, make two identical membranes, and probe one with myc and another with flag antibody.

4. When you run the IPed protein, on the side, run a small measure of your original lysate (un-IPed) as an input control.

5. Once you have seen that myc can pull down flag, and flag can pull down myc-tagged protein, you have proved the interaction in an overexpression system.

6. Next step is to prove it with endogenous proteins. For this, you will need specific antibodies to proteins X and Y.

7. See links for general ideas about immunoprecipitation protocols.

Edited by cellcounter, 03 August 2009 - 08:20 AM.

[henry-fan]

cellcounter, on Aug 3 2009, 06:56 AM, said:

1. Transfect cells with two constructs (test), cons X and cons Y only (negative controls), and untransfected cells (negative control).

2. Lyse, divide each lysate in two aliquots. IP each aliquot with either myc or flag antibody.

3. Run the IPed protein on SDS-PAGE, make two identical membranes, and probe one with myc and another with flag antibody.

4. When you run the IPed protein, on the side, run a small measure of your original lysate (un-IPed) as an input control.

5. Once you have seen that myc can pull down flag, and flag can pull down myc-tagged protein, you have proved the interaction in an overexpression system.

6. Next step is to prove it with endogenous proteins. For this, you will need specific antibodies to proteins X and Y.

7. See links for general ideas about immunoprecipitation protocols.

   Thank  cellcounter,
      other people suggest me to run un-IPed lysate before do IP,  to see if there are x or y protein exist or not, by using flag or myc antibody.
      which way is better , making two identical membranes and probing one with myc and another with flag antibody, or making one membrane and probing by myc antibody first then stripping blot ,probing by flag antibody ?

[cellcounter]

henry-fan, on Aug 3 2009, 08:32 PM, said:

cellcounter, on Aug 3 2009, 06:56 AM, said:

1. Transfect cells with two constructs (test), cons X and cons Y only (negative controls), and untransfected cells (negative control).

2. Lyse, divide each lysate in two aliquots. IP each aliquot with either myc or flag antibody.

3. Run the IPed protein on SDS-PAGE, make two identical membranes, and probe one with myc and another with flag antibody.

4. When you run the IPed protein, on the side, run a small measure of your original lysate (un-IPed) as an input control.

5. Once you have seen that myc can pull down flag, and flag can pull down myc-tagged protein, you have proved the interaction in an overexpression system.

6. Next step is to prove it with endogenous proteins. For this, you will need specific antibodies to proteins X and Y.

7. See links for general ideas about immunoprecipitation protocols.

   Thank  cellcounter,
      other people suggest me to run un-IPed lysate before do IP,  to see if there are x or y protein exist or not, by using flag or myc antibody.
      which way is better , making two identical membranes and probing one with myc and another with flag antibody, or making one membrane and probing by myc antibody first then stripping blot ,probing by flag antibody ?

Doing an input western first is a good idea, but if you have already over-expressed these proteins in these cells and checked by western, you don't have to do it separately.

You just run all your inputs (4 un-IPed samples), and all IPed samples (4+4) in two identical membranes. That is the easiest and most reliable expt design. You can do a lot of variations here, for example, you can also have IgG IP control lanes (4) for each sample. Not essential if you have other controls well done, and if expt gives unambiguous results.

I prefer duplicate membranes, because membrane stripping may be overdone or underdone and may throw you in confusion with no bands or inappropriate bands showing up from previous western, respectively.

[henry-fan]

cellcounter, on Aug 4 2009, 06:18 AM, said:

henry-fan, on Aug 3 2009, 08:32 PM, said:

cellcounter, on Aug 3 2009, 06:56 AM, said:

1. Transfect cells with two constructs (test), cons X and cons Y only (negative controls), and untransfected cells (negative control).

2. Lyse, divide each lysate in two aliquots. IP each aliquot with either myc or flag antibody.

3. Run the IPed protein on SDS-PAGE, make two identical membranes, and probe one with myc and another with flag antibody.

4. When you run the IPed protein, on the side, run a small measure of your original lysate (un-IPed) as an input control.

5. Once you have seen that myc can pull down flag, and flag can pull down myc-tagged protein, you have proved the interaction in an overexpression system.

6. Next step is to prove it with endogenous proteins. For this, you will need specific antibodies to proteins X and Y.

7. See links for general ideas about immunoprecipitation protocols.

   Thank  cellcounter,
      other people suggest me to run un-IPed lysate before do IP,  to see if there are x or y protein exist or not, by using flag or myc antibody.
      which way is better , making two identical membranes and probing one with myc and another with flag antibody, or making one membrane and probing by myc antibody first then stripping blot ,probing by flag antibody ?

Doing an input western first is a good idea, but if you have already over-expressed these proteins in these cells and checked by western, you don't have to do it separately.

You just run all your inputs (4 un-IPed samples), and all IPed samples (4+4) in two identical membranes. That is the easiest and most reliable expt design. You can do a lot of variations here, for example, you can also have IgG IP control lanes (4) for each sample. Not essential if you have other controls well done, and if expt gives unambiguous results.

I prefer duplicate membranes, because membrane stripping may be overdone or underdone and may throw you in confusion with no bands or inappropriate bands showing up from previous western, respectively.

thanks , I will try two membranes  first