Dear Kishore,

How exactly did you isolate the chloroplasts?

Did you dark adapt deveined leaf segments in buffer at 4oC prior to isolation of chloroplasts? Did you break the cells of leaves gently in buffer at 4oC?

In general nucleus (in various Nicotiana species) is much larger (may be over four to eight times) than chloroplasts and you can seperate chloroplasts from nuclei at low centrifugal force. If you are getting nuclear contamination, it could be due to harsh homogenization.  

Thank u sir for your kind reply.

i kept the plants in dark adaptation for 48 hours followed by gentle homogenization at 4 degree. could u suggest me what is the appropriate RPM cor collecting chloroplast fraction over percoll gradient? 

Dear Kishore,

For isolation of chloroplasts, it is not advisable to keep plants for such a long duration of 48 h in dark. Why did you dark adapt plants for 48 h? We have been using sucrose gradient. We carefully lay chloroplast suspension on the surface and centrufuge at a speed of over 12000-16000 rpm. I am sure you can dod the same with percol gradient as well. You need to collect intact chloroplast layer (we prefer using micropipette - cut the tip tips so that you can collect dense suspension with ease) carefully. You can also get a seperate layer of broken chloroplasts/thylakoids. You can test intactness of chloroplasts using standard protocol.

If you can disclose, what exactly is the aim/goal for getting isolated intact chloroplasts, I may be able to further comment.

Thank you sir.

i want to study the chloroplast proteome which requires a chloroplast fraction devoid of other organelleles..