Thermo Fisher
Mouse anti-PSD95
MA1-046
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Stress in vivo, and CRH application in vitro, cause dendritic spine loss in hippocampal pyramidal neurons. This loss of spines, and subsequent synapses, is likely the underlying mechanism through which severe stress disrupts learning and memory. CRH, in combination with neuronal activity and NMDA receptor activation, recruits the actin destabilizing protein, calpain, to break down the actin cytoskeleton resulting in spine loss. Identifying the downstream signaling from CRH receptors could reveal potential therapeutic targets for diseases such as post-traumatic stress disorder (PTSD), anxiety, and depression.
Western Blot
Whole hippocampal cell lysate
1:2000
10% milk in PBS-Tween
1:10,000
None
Thermo Fisher ECL Kit CAT#PI80196
Performed a co-immunoprecipitation with a CRH receptor type 1 and ionotropic glutamate receptors. I used PSD95 as a positive control for the WB and the PSD95 antibody produced two bands. One bad was at the proper weight, 95kDa and the second band was just under 75kDa.Used ECL kit to visualize and developed on Amersham Hyperfilm ECL (GE Healthcare CAT#28906837). Developing times: 12 mins, 10 mins, 8 mins, 4mins, and 2mins with 8 mins exposure being perfect.
Not too many unspecific bands
Extra band at ~75kDa might be a cleavage product
Good antibody to try for WB for a synaptic marker. Very reliable and specific.