Western blot http://westernblot.org Western blot Wed, 11 Apr 2012 13:45:20 +0000 en hourly 1 http://wordpress.org/?v=3.3.2 Western blot results differing with same protein sample http://westernblot.org/2012/04/western-blot-results-differing-with-same-protein-sample/ http://westernblot.org/2012/04/western-blot-results-differing-with-same-protein-sample/#comments Wed, 11 Apr 2012 13:37:36 +0000 jrustybishop@gmail.com http://westernblot.org/?p=318 This weeks Western blot troubleshooting question

From Nick: “Hi! I tried to repeat a western blot from the same protein samples following the same protocol, but the result turned out to be totally different from that of the last time (1st time, down-regulated; 2nd time, no differ; 3rd time, no differ). All bands look pretty without background. I am really confused. Some one argued problems in procedure of ECL developing.

In my protocol, membrane covered with sufficient ECL substrate (Bio-Rad) was incubated for 10min at RT. After that, membrane was grasped vertically by a tweezer to let liquid go off naturally, which took about 15-20 seconds. The membrane was packed in plastic wrap and exposed to X-Ray film for 30s-5min. What do you think? Are there any other reasons for the paradox results?

Looking forward to  your reply. If you need more details, let me know please.

Best wished to you!”

Our Answer – Hi Nick.  That is strange indeed.  If I understand your question correctly, your protein of interest appeared to be down-regulated the first time you did your blot.  Then you wisely repeated the experiment 2 more times and saw no down regulation.

My experience tells me that the simplest explanation is the most likely.  Therefore, I suggest the problem is not in your ECL protocol rather, the results are exactly what you described.  The first time based on the distribution of proteins within the sample you pipetted into the gel/western blot less of your protein of interest went into the gel.  You got a result that was interesting.  After repeating, the western blot with the sample 2 more times the protein no longer seemed to be down-regulated.

The key here to me is that the result the first time is the anomaly not times 2 and 3.Thus I would conclude, with an N=3 that the protein is not down-regulated in that particular sample.

To prove this to your self, you will need to repeat the experiment from the start and do 3 more blots.

To prove to your self its not the ECL you could do a quick dotblot with a known amount of diluted protein using the dotblot protocol described here.

Comments? Ideas?

 

 

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