Ab33655
Manufactured by Abcam
Sourced in United States
Ab33655 is a monoclonal antibody that targets a specific protein. It is designed for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Phospho protease inhibitor cocktail, by Merck Group (2 mentions)
Abn1381, by Abcam (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab108383, by Abcam (1 mentions)
Anti mouse igg hrp conjugate, by Bio-Rad (1 mentions)
Anti rabbit igg hrp conjugate, by Thermo Fisher Scientific (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Ab18987, by Abcam (1 mentions)
Spectra multicolor broad range protein ladder, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Pi 1000, by Vector Laboratories (1 mentions)
Bdnf n 20, by Santa Cruz Biotechnology (1 mentions)
Sc 546, by Santa Cruz Biotechnology (1 mentions)
Nupage novex, by Thermo Fisher Scientific (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Alliance western blot imaging system, by Uvitec (1 mentions)
Ab5316, by Abcam (1 mentions)
7 protocols using ab33655
1
Quantifying BDNF, TrkB, and TrkC Signaling
2
Peptide-Membrane Binding Assay
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The D5 peptide (10 µg and 20 µg) and H4 peptide (10 µg and 20 µg) were attached to a nitrocellulose membrane by applying it drop by drop. The membrane was blocked with 5% nonfat dry-milk solution in TBT-T for 1h and then washed with TBT-T. Membranes were incubated with 1 mg/mL Hc-TeNT for 1h, and afterward, primary antibodies diluted in a BSA 5% solution were added o/n at 4 °C. Antibodies used were mouse anti-polyhystidine (1:1000; 27-471001; Amersham Pharmacia Biotech, Buckinghamshire, UK), rabbit anti-TrkB (1:1000; ab33655; Abcam, Cambridge, UK) and mouse anti-β-tubulin (1:10,000; 556321; BD Biosciences). Secondary antibodies used were anti-rabbit IgG HRP conjugate (Thermo Scientific), and anti-mouse IgG HRP conjugate (Bio-Rad Laboratories, Hercules, CA, USA) diluted 1:3000 in TBS-T plus 5% nonfat dry-milk. Membranes were developed using a 1:1 chemoluminiscent mixture [1 M Tris-HCl pH 8.5, 0.5 M luminol, 79.2 mM p-coumaricacid: 1 M Tris-HCl pH 8.5, 8.8 M H2O2], and exposed to ECL film membranes (Amersham Pharmacia Biotech Buckinghamshire, UK). Analysis of the resulting dots was performed using Quantity One (Bio-Rad Laboratories, Hercules, CA, USA).
3
Immunohistochemistry and Western Blot Analysis of TrkB and Related Proteins
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For TrkB.T1 IHC, the generated mouse TrkB.T1 antibody was used (1.3 mg/ml) at 1:20 and recombinant rabbit fusion SPEH1_D12 (11.5 mg/ml) was used at 1:500 and TrkB kinase antibody (abcam #ab18987; lot: GR3280550-2) at 1:250. For western blot analyses of human brain, mouse brain, and 3T3-Tv-a cells, commercial antibodies were used according to manufacturer specifications and bands were confirmed by size using Spectra™ Multicolor Broad Range Protein Ladder (ThermoScientific™ catalog # 26634; lot: 00784968): pSTAT3 (Tyr705, Cell Signaling #9145; lot: 22 at 1:1000), pAkt (Ser473, Cell Signaling #4060; lot: 23 at 1:1000), pERK (Cell Signaling phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) XP® #4370; lot: 12 at 1:2000), β-actin (Sigma #AB1978 Clone AC-15; lot: 021M4821 at 1:10,000), pS6 ribosomal protein (Ser235/236, Cell Signaling #4858; lot: 16 at 1:1000), TrkB (Millipore #07-225; lot: 2187222 and lot: 3277578 @ 1:3000), TrkB (abcam #ab33655; lot: GR266297-1 at 1:1000), TrkB (Abcam #ab18987; lot: GR3280550-2 at 1:2000), pPDGFR β (Y1021; Abcam #ab62437; lot: GR38791-13 at 1:200), PDGFR β (Cell Signaling #3169; lot: 13 at 1:800), ERBB3 (Cell Signaling #12708; lot: 4 at 1:1000), NRG2 (Abcam #ab220615; lot: GR3181158-4 at 1:200), vinculin (Sigma-Aldrich, Cat. V9131; lot: 118M4777V at 1:10.000) and added in 5% BSA/TBST overnight at 4 °C.
4
TrkB Activation Assay in NIH-3T3 Cells
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NIH-3T3-TrkB-expressing cells were plated in 12-well plates at 100,000 cells/well and the following day were serum deprived for 6 h. Compound treatments to assess TrkB activation were performed at 1 μM for 20 min, while BDNF was used as a positive control at 500 ng/mL. Lysis was performed using a lysis buffer on ice (Thermo Scientific Pierce IP Lysis Buffer) with a Millipore phospho-protease inhibitor cocktail by Millipore for 10 min. After adding the loading buffer (5× Laemni) to 25 μg of the protein sample, samples were incubated at 95 °C for 5 min and subjected to SDS-PAGE. Nitrocellulose membrane transfer was performed at 350 mA for 2 h, followed by blocking at room temperature with 5% bovine serum albumin (BSA) for 1 h and primary antibody incubation in blocking solutions at 4 °C overnight and detection with HRP-conjugated secondary antibodies. ECL solution was used to detect chemiluminescence. Primary antibodies used are Phospho-TrkB (Tyr816) Millipore # ABN1381 and Anti-TrkB Abcam #ab33655, β-Actin.
5
TrkB Phosphorylation Assay Protocol
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Cells were plated in 12 well plates, at a density of 100,000 cells per well, with 6 h of serum deprivation carried out on the following day. To assess the effect of compounds on TrkB phosphorylation, 20-min treatments were applied, using either BDNF at 500 ng/mL (Peprotech 450-02) (positive control) or compounds at 1 μM.
Cell lysis for 10 min was carried out on ice using the Pierce IP Lysis Buffer by Thermo Scientific and a phospho-protease inhibitor cocktail by Millipore. After adding loading buffer (5 × Laemni), 25 μg from each protein sample were incubated for 5 min at 95°C and subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane at 350 mA for 2 h. Membrane blocking was performed using 5% bovine serum albumin (BSA) at room temperature for 1 h, before adding the primary antibodies to the blocking solution at 4°C overnight. HRP-conjugated secondary antibodies were used for the detection of chemiluminescence with ECL solution. Primary antibodies: Phospho-TrkB (Tyr816) Millipore # ABN1381, and Anti-TrkB Abcam #ab33655.
Cell lysis for 10 min was carried out on ice using the Pierce IP Lysis Buffer by Thermo Scientific and a phospho-protease inhibitor cocktail by Millipore. After adding loading buffer (5 × Laemni), 25 μg from each protein sample were incubated for 5 min at 95°C and subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane at 350 mA for 2 h. Membrane blocking was performed using 5% bovine serum albumin (BSA) at room temperature for 1 h, before adding the primary antibodies to the blocking solution at 4°C overnight. HRP-conjugated secondary antibodies were used for the detection of chemiluminescence with ECL solution. Primary antibodies: Phospho-TrkB (Tyr816) Millipore # ABN1381, and Anti-TrkB Abcam #ab33655.
6
Retinal Protein Extraction and Western Blot
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Retinas were excised from the eye cup immediately after death, and tissue was snap-frozen on dry ice before lysing using a Lysis-M reagent containing cOmplete Mini Protease Inhibitor (Roche) and phosphatase inhibitors (Thermo Fisher Scientific). Following 20-min homogenization, tissue was centrifuged at 13,000 rpm for 10 min to isolate the soluble cell extract. Protein concentration was determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific), and equal quantities of protein were loaded onto 10% or 4 to 12% bis-tris gels (NuPAGE Novex, Thermo Fisher Scientific). Membranes were blocked in 5% dried skimmed milk in PBS with 0.2% Tween 20 (Sigma-Aldrich) for 60 min and then incubated overnight at 4°C in primary antibody (BDNF N-20, rabbit, 1:200, sc-546, Santa Cruz Biotechnology; TrkB, rabbit, 1:500, ab33655, Abcam; β-actin, rabbit, 1:1000, 4967, Cell Signaling Technology). Primary antibodies were visualized with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:8000; PI-1000, Vector Laboratories) and signal detection using ECL Prime (GE Healthcare) and an Alliance Western blot imaging system (UVItec Ltd.).
7
Western Blot Analysis of Synaptic Proteins
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