TrkB [p Tyr707, p Tyr706] Antibody - BSA Free

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• TrkB [p Tyr706] Antibody

  Name: francesca mottarlini

  Application: Western Blot

  Sample Tested: Rat brain tissue, Rat brain synaptosomal (P2) fraction and Rat brain lysate (ventral hippocampus)

  Species: Rat

  Verified Customer | Posted 02/19/2019

  F1 pTrkBTyr706 mPFC HOMO, P2. 145 kDa F2 pTrkBTyr706 95 kDa F3 total TrkB Primary TrkB ab #4603 Cell signalling F4 beta-Actin F5 pTrkBTyr706 Hipp HOMO 145kDa, 95kDa F6 total TrkB,145kDa and 95kDa F7 beta-Actin

  I’m writing to inform you about the Western Blot trial that I have done regarding the pTyr706 NBP2-54764; antibody that I bought Order n° 1334 15/11/2018 and that was untested for WB analysis. First of all, in the datasheet that you provide, bands of trkB are expected at 95kDa. However, we know from the literature that trkB receptor is present in two forms: the full length that weights 145 kDa and the truncated one that weights 95 kDa. As you can see from the blots attached, I effectively obtained with your antibody (against the phosphorilated form) and with an antibody against the total form of trkB (cell signaling #4603) two different bands at these specific molecular weights. The test was conducted on rat brain prefrontal cortex tissue extracts (Figure 1-4), and on rat brain hippocampus (Figure 5-6) obtained following the below-mentioned protocol. Prefrontal cortex (Pfc) tissues were processed following a classic protein extraction method done at room temperature, which allow us to obtain aliquots of whole homogenate (HOMO), nuclei (P1), cytosol (S2) and a crude membrane fractions (P2). 1. Pfc tissues were homogenized in a glass-glass potter in a homogenization buffer (0,32 M sucrose, 0,1 mM PMSF, 1 mM Hepes, 0,1 mM EGTA pH 7,4, 1X Complete (protease inhibitor cocktail), 1X phosphatase inhibitor cocktail). From that step we kept an aliquot of homogenate, we sonicated it and maintained at -20°C for following WB analysis. 2. The left homogenate was moved on through the extraction and centrifuged 10 minutes at 4°C at 1000g. 3. The obtained surnatant S1 was again centrifuged 15 minutes at 4°C at 9000g to obtain the P2 and the S2, while the pellet was the P1 fraction that was resuspended in a buffer solution (20 mM Hepes, 0,1 mM DTT (ditiotreitol), 0,1 mM, EGTA pH 7,4, 1X Complete, 1X phosphatase inhibitor cocktail). The obtained P2 was as for the P1 resuspended in the same previous buffer. Protein extracts were separated by polyacrylamide SDS/PAGE and transferred to nitrocellulose membranes. Blots were immunostained overnight at 4°C with the specific primary antibodies (see figures for specifics). After washing, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibody (anti-mouse, anti-rabbit). After secondary antibody incubations, membranes were washed and finally incubated with ECL detection reagent. Western blot-1: - Figure1: blot of pTrkB Tyr706 measured in rat brain extracts of Prefrontal cortex in the whole homogenate (HOMO) and crude synaptosomal fraction (P2). We revealed one band at 145 kDa, molecular weight of Full length TrkB. - Figure2: blot of pTrkB Tyr706 measured in rat brain extracts of Prefrontal cortex in the whole homogenate (HOMO) and crude synaptosomal fraction (P2). We revealed one band at 95 kDa, molecular weight of truncated TrkB. - Figure3: blot of total TrkB measured in rat brain extracts of Prefrontal cortex in the whole homogenate (HOMO) and crude synaptosomal fraction (P2). Primary TrkB antibody (#4603) Cell signalling - Figure4: blot of beta-Actin (Molecular weight 43 kDa) - Prefrontal cortex Western blot-2: - Figure5: blot of pTrkB Tyr706 measured in rat brain extracts of hippocampus in the whole homogenate (HOMO). We revealed two bands: one at 145 kDa molecular weight of the TrkB full length and one at 95 kDa (truncated TrkB). - Figure6: blot of total TrkB measured in rat brain extracts of hippocampus in the whole homogenate (HOMO). We revealed two bands: one at 145 kDa molecular weight of the TrkB full length and one at 95 kDa (truncated TrkB). - Figure7: blot of beta-Actin (Molecular weight 43 kDa) - Hippocampus

  

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