NMDA receptor expression, unable to resolve receptor subunits - (May/25/2007 )
Hi , can anyone help me in resolving NMDA receptor subunits from rat brain through SDS PAGE. I had tried variuos protocols but unable to get the bands of NR1.NR2A-B, subunits , as these bands are in the range of 107-180 KDa range and I am not getting any such band in upper section of gel. The lower portion is resolving successfully ( from 60-30 Kda). i am unable to find where is the problem whether in brain homogenate preperation (supernatant of brain parts homogenate in tris lysis buffer containing protease inhibitors after centrifugation at *800g ) or other wise in Gel. all suggestion are welcome. I posted this already on neuroscience section but only one reply i got and that too on my email.as i am a pharmacologist and dont have much idea of mol bio techniques thus i welcome every bit of your's valuable suggestions.
I'm not sure if I will be trully helpful since I don't know the protocol you are using but here are some suggestions-questions
1. Is the concetration of your gel right? I mean for the bands you are looking for (107-180) a gel of 5-6% on acrylamide would be more appropriate.
2. Let the electroforesis run longer. Even if the lower bands "get out".
3. Do you have a sample that you can use as a positive control, that is a sample that you are sure that has these subunits? Are you sure that these subunits are expressed in your sample?
Hope you have better results next time.
Dear Charis, my protocol includes removal of rat brain at 40 C and remonig its various parts ( cortex hippocampus,straitum) ,weighing them and homogenizing these seperately in buffer consisting of ( 50 mM MOPS, 100 mM KCl,320 mM sucrose,0.5 mM MgCl2, 0.2mM DTT ,phosphatase and protease inhibitors( i`m having only few PMSF,Sod. orthovandate,EDTA,EGTA,).THE homogenizing is done using polytron and the homogenates are centrifuged at 800 X g for 10 min at 40 C , supernatant collected and protein estimated .this sample was uesd to be run in SDS PAGE (8 %) , as you suggested i`ll run at 6% and for longer periods.I`m not having any positive control for that subunit but can you suggest me some even higher mol weight proteins so that it could be used to detect whether the GEL is resolving it or not. The NMDA receptor is involved in memory and cogizance function thus they are definitely expressed and highly in cortex, under certain pathological conditions there expression is increased. if there is any flaw in protcol just point it out. thanks for your suggestions, i`ll apply these and then reply you with outcome.
I cross-checked your protocol fot homogenization with my supervisor (just to be sure because she's quite experienced)and she said that it's OK.
nNOS (neuronal nitric oxide synthase) is an enzyme with a molecular weight of 300KDa, maybe you can use that or check for another enzyme preferably (not a receptor) that has more appropriate size.
Maybe try to add P.I. coctail as well as PMSF? (if you can find any...try asking for a little from another lab just to check if the absence of it has to do with your problem).
Good luck again!