To confirm the presence of rrβ-NGF, the dialyzed protein was subjected to TCA-acetone precipitation and then denatured in loading buffer (0.312 M Tris-HCl, 10% SDS, 25% 2-mercaptoethanol, 0.01% bromophenol blue, 50% glycerol) at 95°C. The denatured protein was then loaded on 12% SDS–PAGE gels which were run at 90 V for 2 h in duplicate. One gel was stained with Coomassie brilliant blue G-250 (Sigma Aldrich, Missouri, USA) and the other was used to transfer the protein to a nitrocellulose membrane (Ammersham Hybond ECL Nitrocellulose Membrane, GE Healthcare Life Science, Barcelona, Spain) (80 mA per membrane for 80 min). The membrane was blocked for 1 h with Odyssey blocking buffer (LI-COR Biosciences, Nebraska, USA), and then incubated at 4°C overnight with 0.1 μg/mL of goat anti-NGF antibody (N8773, Sigma-Aldrich) in blocking buffer with 0.1% Tween 20. After 6 washes, membranes were incubated at room temperature (RT) for 1 h with secondary antibody (IRDye 800CW Donkey anti-goat IgG (H + L), LI-COR Biosciences, Nebraska, USA). Finally, after additional washes, the membranes were scanned with an Odyssey fluorescence scanner (LI-COR Bioscience, Nebraska, USA).
Irdye 800cw donkey anti goat igg h l
Manufactured by LI COR
Sourced in United States
IRDye 800CW Donkey anti-goat IgG (H + L) is a secondary antibody conjugated with the IRDye 800CW fluorescent dye. It is designed to detect and quantify goat primary antibodies in various applications such as Western blotting, immunohistochemistry, and in-cell Western assays.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (2 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Odyssey fluorescence scanner, by LI COR (1 mentions)
Hybond, by Cytiva (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti phosphoerbb3, by Cell Signaling Technology (1 mentions)
Irdye 680lt donkey anti mouse igg h l, by LI COR (1 mentions)
Anti erbb3, by Cell Signaling Technology (1 mentions)
Anti cd74, by Abcam (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
β actin hrp, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti hsp90, by Cell Signaling Technology (1 mentions)
Irdye 680rd donkey anti rabbit igg, by LI COR (1 mentions)
Immobilon fl transfer pvdf membranes, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Odyssey clx infrared imager system, by LI COR (1 mentions)
4 protocols using irdye 800cw donkey anti goat igg h l
1
Western Blot Validation of Recombinant NGF
2
Immunoblotting of NRG1 and Downstream Signaling
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Immunoblotting was performed as described previously (1 (link)). The following antibodies were used: anti-NRG1 β1 (R&D, AF-396-NA), anti-CD74 (Abcam, ab22604), anti-HSP90 (Cell Signaling Technology, No. 4877), anti-ERBB3 (Cell Signaling Technology, No. 4754), anti-phosphoERBB3 (Cell Signaling Technology, No. 4791), ERK1/2 (Cell Signaling Technology, No. 9102), anti-phosphoERK1/2 (Cell Signaling Technology, No. 9106), anti-AKT (Cell Signaling Technology, No. 9272), anti-phosphoAKT (Cell Signaling Technology, No. 9271), and β-actin-HRP (Santa Cruz Biotechnology, sc-47778). Secondary antibodies were IRDye800CW donkey anti-goat IgG (H+L; Licor, 925–32214), IRDye 680LT donkey anti-mouse IgG (H+L; Licor, 925–68022), and IRDye 800CW goat anti-rabbit IgG (H+L; Licor, 926–32211). Fluorescence detection was performed on Odyssey CLx Imaging System (Licor).
3
Cerebellar Protein Expression in Dyt1 KI Mice
4
Western Blot Analysis of Benzonase-Eluted Proteins
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Following the described RAP-MS procedure, we saved 5% of benzonase eluted proteins for western blot analysis. We added NuPAGE LDS Sample Buffer (Thermo Fisher Scientifc) and incubated samples for 3 min incubation at 95°C. Proteins were resolved by SDS-PAGE using NuPAGE 4-12% Bis-Tris-HCl Gels (Thermo Fisher Scientific) at 200 V for 1 h, followed by transfer to a nitrocellulose membrane using the iBlot Dry Blotting System (Thermo Fisher Scientific). Western blots were performed using the iBind Western System (Thermo Fisher Scientific). For protein detection, we used the following primary antibodies: Nucleoprotein -Abcam #ab272852, POP1 -Proteintech #12029-1-AP. We used the following secondary antibodies: IRDye 800CW Goat anti-Rabbit IgG (H + L) (LI-COR), IRDye 800CW Donkey anti-Goat IgG (H + L) (LI-COR). For visualization of bands, we used the Odyssey Clx infrared imager system (LI-COR).
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