Immunoprecipitation – Protocol

· Lyse cells / tissue in: 20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM EDTA ( or 1,7 mM CaCl2) , 0,5% NP-40 – 1% NP40 + Protease inhibitors and use a Douncer (For whole cell lysate use sonicator)

· Incubate crude lysate on ice for 30 min

· Centrifuge at 12.000 x g for 15 min 4°C

· In the meantime wash 40 µl Protein-G-Agarose (or A) in 1000 µl PBS for 3 times, centrifuge at 2000 x g for 2 min after every washing step

· Take sample of cell lysate (supernatant; discard cell pellet) (e.g. 20- 40 µl) as a positive control for IP

· Pre-incubate the cleared supernatant with Protein-G-Agarose for 1 h at 4°C (Pre-clearing)

· Take cleared lysate to new tube and add 0,5 -1 µl crude serum or 5-10 µg of commercial antibody for 3 h at 4°C and 0.5-1 µl non-specific serum of same species (Antibody pre-incubation)

· In the meantime wash new 40 µl Protein-G-Agarose (or A) in 1000 µl PBS for 3 times, centrifuge at 2300 x g for 5 min after every washing step

· Add pre-incubated supernatant to new Protein-G-Agarose over night at 4°C

On the next day:

· Wash IP 5 times with lysis buffer (quite stringent washing, reduce NP40 if required) and centrifuge for 5 min at 2300 x g after every washing step

· Wash IP 1 time with PBS

· Take up pellet in wanted volume

· Add β-Mercaptoethanol and loading buffer to your samples and boil for 5 min at 100°C

· Centrifuge 5 min 13.000 x g

· Load samples on SDS-PAGE

BTW. A slightly modified version of my protocol I use for cell culture supernatants as well. Works really good!

You can't expect an answer for a vague question such as this, since there is no information on the type of protein you are seeking to isolate, the host organism/cell it was obtained from, etc. Please do more research on Western Blotting and IP techniques, rather than expect an answer to a generic question of this nature.

This is an extracellular protein majorly localized in epithelial basement menbranes. The extract is from mouse new born skin which we usually expect to see a high expression of the protein of interest but someone no signal was shown on several blots from the extract. I have the impression that perhaps the extract is too diluted and wanted to perform immunoprecipitation to somehow concentrate the protein for easy detection on blot.

There are many (possibly an uncountable number of) reasons why you never got any signal on the Western Blot. Some of these include the membrane you used for the blot (NC vs PVDF, which have different affinities for different proteins), the expression level of the protein you are seeking to isolate, the method you used to extract the protein (are you absolutely certain that you have isolated the correct protein, and did you verify this using sequencing, or simply SDS-PAGE?), the buffers you used throughout the experimental protocol, etc. All of these factors are crucial, and none of these are constants since they vary for different proteins that you may want to isolate. This is one of the hurdles of proteomic research, which everyone doing research in this field has to encounter and resolve.

Thanks for your response, the problem is not the membrane as we can easily detect signal on the recombinant protein with the same NC membrane. As i said we expect to see a high expression level according to previous research works and immnunohistochemistry analysis on tissue sections. The extraction was done sequencially using differrent buffers ranging from mild to denaturing conditions and none gave a signal. And if I may ask, can you further expatiate on using sequencing to verifiy my protein of interest?

Thanks

Thanks for your response as well. However, there seems to be some anomaly here - earlier, you mentioned that you can easily detect the signal of the recombinant protein with the same NC membrane, but you were expecting high expression levels due to previous studies claiming as such. Subsequently, you mentioned that the extraction was done sequentially using differrent buffers ranging from mild to denaturing conditions and none gave a signal.

The question now is: what exactly is the key issue here - are you able to get a signal on the Western Blot (except that it is a weak one & you are seeking to boost it) or are you not even able to get any signal at all?

The recombinant protein was easily detected by my antibody which might implies that the antibody is working quite well. However there is no siganl at all on this skin new born tissue extracts with varying buffer conditions. We expect to see a high level of expression of the protein at this stage of skin development according to previous studies.

It's likely that your extraction buffer might have something to do with your failure to get any signal - did you add a suitable protease inhibitor (e.g. DMSO) to inhibit any proteases produced during cell lysis and extraction of the protein?

Immunoprecipitation – Protocol

· Lyse cells / tissue in: 20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM EDTA ( or 1,7 mM CaCl2) , 0,5% NP-40 – 1% NP40 + Protease inhibitors and use a Douncer (For whole cell lysate use sonicator)

· Incubate crude lysate on ice for 30 min

· Centrifuge at 12.000 x g for 15 min 4°C

· In the meantime wash 40 µl Protein-G-Agarose (or A) in 1000 µl PBS for 3 times, centrifuge at 2000 x g for 2 min after every washing step

· Take sample of cell lysate (supernatant; discard cell pellet) (e.g. 20- 40 µl) as a positive control for IP

· Pre-incubate the cleared supernatant with Protein-G-Agarose for 1 h at 4°C (Pre-clearing)

· Take cleared lysate to new tube and add 0,5 -1 µl crude serum or 5-10 µg of commercial antibody for 3 h at 4°C and 0.5-1 µl non-specific serum of same species (Antibody pre-incubation)

· In the meantime wash new 40 µl Protein-G-Agarose (or A) in 1000 µl PBS for 3 times, centrifuge at 2300 x g for 5 min after every washing step

· Add pre-incubated supernatant to new Protein-G-Agarose over night at 4°C

On the next day:

· Wash IP 5 times with lysis buffer (quite stringent washing, reduce NP40 if required) and centrifuge for 5 min at 2300 x g after every washing step

· Wash IP 1 time with PBS

· Take up pellet in wanted volume

· Add β-Mercaptoethanol and loading buffer to your samples and boil for 5 min at 100°C

· Centrifuge 5 min 13.000 x g

· Load samples on SDS-PAGE

BTW. A slightly modified version of my protocol I use for cell culture supernatants as well. Works really good!

Thanks Julian