I have recently started my first post-doc, switching from a pharmacology lab to a neurology lab, and am in the process of optimizing lysate generation from mouse nerve tissue as the current methods I feel are sub-optimal.
One of the main problems is that there is a low level of protein from within this tissue type therefor it is essential to maximise yield from our samples.
Original protocol used in the lab:
-Nerve (collected in a 1.5ml tube) is pulverised on dry ice using blue pestle (see image below).
-100 ul of TPER lysis buffer (containing protease inhibitor) is added and tissue is homogenized using the blue pestle (small volume to maximize protein conc).
- Sample is sonicated for 5 sec (30% amp) x2 (this generally generates a foamy sample)(users tend not to keep on ice as they try to keep the probe in the sample).
- Samples are spun for 20 min at 4C at 16,000 x g, the supernatant is collected and protein determination assessed.
The main issues I have had using the above method is the sonication step. As the protein yield is expected to be low, a minimal amount of lysis buffer is added in order to keep the concentration between 0.5-1.5 mg/ml. However, sonication of a this small volume using the method above leads to foaming and sample loss. I feel this is the reason for the poor quality of several of our immunoblots.
The modifications that I have introduced include, (1) using a 2 ml Douce homogenizer instead of the blue pestles (0.2 ml homogenizers have also been ordered) (2) performing 0.5 sec sonication pulses x3 cycles of the lysate, and (3) persuading our supervisor to buy a sonicator with a cup-horn attachment for indirect sonication (eliminates sample loss and foaming).
With the minor modifications I have made so far, my protein yield has increased nearly 2-fold due to improving homogenization and also, my resulting immunoblots are looking much better.
As we want to standardize this protocol for the lab I was wondering if anyone else had any suggestions/advice that can be introduced to increase protein yield and minimize degradation of sample?
Dear Researcher:
Really I believe that to optimize your nerves preparation you could to add any non ionic detergent as Triton X-100 or Tween 20. I use a Polytron homogenizer soft tissue. Generally I use between 8 and 12 frozen sciatic nerves ciaticos in a volume not greater of 1 ml of ice cold lysis buffer (150 mM NaCl + 50 mM Tris-HCl pH. 8.0 + 5 mM EDTA + a cocktail of protein inhibitor). You can make 20 pulse of 10-20 seconds with intervals of 1 min always on ice. Subsequently spin to 14,000 X g to {PN4° CPN} during 20 min. Taking the sobrenadante for the quantification of proteins. Tris procedure yields sufficient quantity of proteinas for various WB.
I´m not sure if use of detergent would affect your homogenate in accord your objectives.
Best regards
A related question -
Has anyone used a bead homogenizer for extraction of proteins and/or RNA from sciatic nerves? I have a bead-beater instrument and 3.0 mm beads. I've gotten low yields with sciatic nerve, but I'm wondering if that's because I'm using a protocol that is not appropriate. Should I chop up the nerve into small pieces first? Prolong the beating cycle? Any advice would be greatly appreciated.
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