Hi everyone,
I am working on gut cannulation to determine some specific proteins in gut. So I have to collect the gastric fluid as pure as possible for running western blot. But the problem is the fluid is very little, if I feed mouse/rat, then that fluid will be very dirty.
So how can I collect the gastric fluid with the reasonable amount?
How about fasting the animals for a few hours and placing something in their cages that does not allow them to access food but they can smell it.
Just a cruel thought... but it may work.
Or switch to a larger animal.
Thank you so much for your advice, I think it is possible to try. And I am wondering is there any protocol for western blot these fluid? like gastric fluid, jejunum, duodenum fluid... ?
Hi Triet,
we usually make the animal to do an overnight fast for solids. In the morning, you ligate the pylorus of the anesthetized animal and put 500 ul of ice cold normal saline through oral gavage. Now ligate the esophagogastric junction and let the saline in there for 15 mins. Now carefully take the stomach out and collect the fluid. centrifuge it at 10,000 rpm for 10 mins and collect the supernatant. this should have sufficient protein in it, you can do usual BCA and western or you can do TCAA extraction and then a western. hope this was helpful.
shrey
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models