Thanks for your clarification Gaurav. I shall suggest you do the IP like this: I am assuming you have a purified His tagged protein. First test your purified protein with a western blot where you load different amounts of the purified protein and check that it gives one band on a western. As a rule of thumb 5 micro-grams of purified protein should be enough for the first attempt. So use 5 micrograms of your purified protein, 5 micrograms of anti-His antibody and get lysate from 50 ml of yeast culture ( I am assuming its S.cerevisiae) with a final OD of 1.0 and leave it O/N on a end over end rotator in the cold room (4 degree C) . The lysate volume should be no more than 600 microlitres. Next morning add washed protein A beads and rotate for another 4 hours. Finally take the microfuge tubes, spin at 2000 rpm (higher speed shall destroy the beads so be careful) . Store the lysate at 4 degrees and continue with the beads. Wash the beads in the breaking buffer (I use IP 150 for breaking and washing whose composition is IP150 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM MgCl2, 0.1%

NP40. While breaking I add protease inhibitor cocktail tablets and phosphatase inhibitor cocktail tablets to it). Wash for 3 times (washing means turn the tube up and down for one minute), spin down 1 min at 2000 rpm and aspirate the supernatant and then after the final wash make sure to remove as much of the liquid as you can to make the beads dry, resuspend in 20 ul (microlitre) of SDS-PAGE leading buffer. Boil for 10 mins at 100 deg C and load onto a protein gel. Run it, transfer it onto a nitrocellulose membrane, and probe it with the anti-His antibody. If you are lucky you shall see the band of your recombinate protein and some more ones to which your protein may be binding.

There are lots of available protocols. But what exactly are you trying to do? You have a tagged recombinat protein with which you are trying to pull down yeast proteins to see if something interacts? Its not from your question. If you provide some information, there is a good chance I would be able to provide you with a suitable protocol.

thank you !! Jaideep..i have His tag recombinant protein, and i am looking for the interacting protein against the his tag protein..there might be many protein which interacts with my recombinant protein..i am working in yeast model..

i appreciate your suggestion and help..

Thanks for your clarification Gaurav. I shall suggest you do the IP like this: I am assuming you have a purified His tagged protein. First test your purified protein with a western blot where you load different amounts of the purified protein and check that it gives one band on a western. As a rule of thumb 5 micro-grams of purified protein should be enough for the first attempt. So use 5 micrograms of your purified protein, 5 micrograms of anti-His antibody and get lysate from 50 ml of yeast culture ( I am assuming its S.cerevisiae) with a final OD of 1.0 and leave it O/N on a end over end rotator in the cold room (4 degree C) . The lysate volume should be no more than 600 microlitres. Next morning add washed protein A beads and rotate for another 4 hours. Finally take the microfuge tubes, spin at 2000 rpm (higher speed shall destroy the beads so be careful) . Store the lysate at 4 degrees and continue with the beads. Wash the beads in the breaking buffer (I use IP 150 for breaking and washing whose composition is IP150 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM MgCl2, 0.1%

NP40. While breaking I add protease inhibitor cocktail tablets and phosphatase inhibitor cocktail tablets to it). Wash for 3 times (washing means turn the tube up and down for one minute), spin down 1 min at 2000 rpm and aspirate the supernatant and then after the final wash make sure to remove as much of the liquid as you can to make the beads dry, resuspend in 20 ul (microlitre) of SDS-PAGE leading buffer. Boil for 10 mins at 100 deg C and load onto a protein gel. Run it, transfer it onto a nitrocellulose membrane, and probe it with the anti-His antibody. If you are lucky you shall see the band of your recombinate protein and some more ones to which your protein may be binding.

Thanks a lot for your valuable suggestion jaideep. i will try to set the experiment according to your protocol..and get back to you with my results and comments..thanks

However having said all that I would suggest that before you do all that just do another experiment. A far western should be done ebfore you do the IP. Now what is a far western? Run a western blot with lysate from your yeast cells (just like in IP) with a range of total protein amounts (ideally the range should be like 50-500 micrograms of protein). You should have two sets of lanes on your gel. Both sets shall be stransferred to the NC membrane and one set should be incubated first with the his tagged protein, then washed and incubated with anti his anti-body and then proceeded as normal, while the membrane containing the other set should just be incubated with the secondary antibody (and the second membrane is the negative control). This experiment shall give you some idea as to how many proteins are interacting with your recombinant protein and shall help you interpret your IP results!!

Thanks again Jaideep..I have one more query i.e. after overnight incubation of yeast lysate and recombinant protein..then we incubate those in NI-NTA Beads, so during washing should we go for the different concentration of imidazole for washing as in case of purification or just wash with the PBS buffer...

If you are planning to use Ni-NTA-Agarose beads, just follow the manufacturers protocol. Maybe this page shall help you :http://www.piercenet.com/method/his-tagged-proteins

And best of luck!!

Hi Jaideep, 

Would you have suggestions regarding protein concentration to use of  the his-tagged protein for the far westerns?

Thanks!

Sofia