Hello reseachers',
I am trying to get the blot of NLRP3 in liver tissue homogenate of SD and Wistar rat, but i do not have any result. I have used the antibody purchased from santa cruz, novus, CST and abcam. the protocol I have followed is below,
1. one month stored (-20 degree C) liver tissue was homogenised (RIPA buffer containing protease inhibitor )and centriguged at 10000-20000 *g for 10-20 minutes.
2. probe sonicated for 10 seconds for 2 times, protein concerntration was 1-10 mg/ml
3. used 2X sample buffer and 5-50 microlite of sample was loaded and run on 7-10 % acrylamide gel
4. transfered to PVDF/NC membrane
5. Blocked with 3% BSA/5% NFDM for 1-3 hour at room temperature
6. overnight incubated with primary antibody at 1:200 to 1:1000 in TBS/TBST
7. incubated with secondary ab for 1 hour at room temperature (1;20000)
8. visualised with ECL and X-ray with exposure time 1 min-12 hour.
Kindly suggest me.
Thanks in advance.
Hello Durgesh.
Sorry.
It should have been stored at -70C after plunging into LN2 gas to prevent degradation during slow freezing at -20C from warm liver tissues. Frequent opening and closing the door might cause many repeated thawing and freezing process.
Regards.
As Sang noted, it is likely that your liver sample may have degraded NLRP3. This raises the question- did you see any low molecular weight protein bands in lanes that had liver samples? Did the immunoblotting work for a positive control (an extract from THP-1 cells)? If not, there may be other issues. Share data next time to identify the problem. In our hands, the blotting works well for many tissue extracts. We will be happy to share a protocol.
have you read this paper yet?
https://www.nature.com/articles/s41598-017-17634-1
might help, it focuses entirely on detecting NLP3 using different commercial antibodies
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