Trying to run western blot for the protein extracted from mice kidney. Mice induced with 20 mg/kg B.w LPS for 14-18 hr. The dissected kidney frozen in -80. Having trouble by the presence of blood cells in the homogenized kidney. Facing difficultly in detangling protein (I tried 80 degree 2min, 37 degree for 30 min, 100 degree 5 min)
Is there any perfusion method during dissection is available to get rid of RBC from kidney tissue.
Please share the experience in extracting protein from mice kidney. Thank you in advance
You can Wash and centrifuge the homogenized kidney samples by
3X time centrifuge the sample at 8000 RPM at 4 deg celsius for 5 min each.
In between, wash the samples with ICE COLD PBS.
Homogenized samples+ (15-20ml) PBS= centrifuge at 4 deg for 8000 RPM for 5 min 3x time.
This will remove all the RBCs from the samples.
After that, you can prepare samples for running western blotting.
All the best!
Happy Research.
Deepika Subramanyam
You don't necessarily need to perfuse the whole animal. Simply fill a 5ml syringe with pbs, remove the kidney starting with the aorta/renal artery, insert a 25G needle into the artery and gently wash. This way you will have a cleaner sample, saving time.
Thank you so much Tahib Habshi and Marcos Divino Ferreira, information you both shared is very useful. I am going to try both the method and see how it works one for already dissected kidney (stored) and the another for upcoming experiments.
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