Anti darpp 32

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-DARPP-32 is a primary antibody product manufactured by Cell Signaling Technology. It is designed to detect DARPP-32 (Dopamine and cAMP-Regulated Phosphoprotein 32), a protein involved in signal transduction pathways. The antibody can be used in various analytical techniques to study the expression and localization of DARPP-32 in biological samples.

Automatically generated - may contain errors

Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (2 mentions) Anti βiii tubulin, by Abcam (1 mentions) Anti phospho eif2α ser51, by Cell Signaling Technology (1 mentions) Anti gapdh, by Merck Group (1 mentions) Normal goat serum (ngs), by Vector Laboratories (1 mentions) Anti bip, by Merck Group (1 mentions) Anti total eif2α, by Cell Signaling Technology (1 mentions) Goat anti rabbit igg conjugated to cy3, by Jackson ImmunoResearch (1 mentions) Goat anti rabbit and anti mouse igg conjugated to hrp, by Jackson ImmunoResearch (1 mentions) Anti phospho perk, by Cell Signaling Technology (1 mentions) Anti gfp, by Santa Cruz Biotechnology (1 mentions) Anti γh2ax antibody, by Merck Group (1 mentions) Nb100 304, by Novus Biologicals (1 mentions) Anti htt, by Merck Group (1 mentions) Anti gst, by Santa Cruz Biotechnology (1 mentions) Mouse anti β actin, by Merck Group (1 mentions) Anti β actin, by Merck Group (1 mentions) Hrp conjugated secondary antibody, by GE Healthcare (1 mentions) Anti erk, by Cell Signaling Technology (1 mentions) Anti phospho erk, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

DARPP-32 (12 citations) Rabbit anti-DARPP-32 (6 citations) Rabbit anti-DARPP-32 IgG (4 citations)

10 protocols using anti darpp 32

Quantitative Analysis of Neuronal Morphology

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the analysis of neurite morphology, treatments were performed the next day after seeding the neurons. Treatments were added to the fresh medium when the medium was partially replaced. MSN cultures were fixed with 4% paraformaldehyde after 10 days in vitro, after which they were permeabilizated with 0.1% Triton-X100 and blocked with 1% bovine serum albumin (BSA) before labelled with anti-DARPP-32 (1:400 Cell Signaling Technology, Danvers, MA, USA) to identify MSNs. Analysis of neuronal morphology was studied after double labelling with anti-βIII tubulin (1:1500; chicken ab41489, AbCam, Cambridge, UK). Secondary antibodies used to visualize labelling neurons were fluorescent Alexa antibodies (1:500; Thermofisher, Cambridge, UK, A-21206 and A-11042). Labelled neurons were visualized using a Zeiss LSM710 confocal microscope.
Neurite length was assessed using Fiji (ImageJ) software with the semiautomated plugin Simple Neurite Tracer [15 (link)]. The mean and standard errors of the measurements from at least three independent experiments were plotted.

Carriba P, & Davies A.M. (2021). Signalling Pathways Mediating the Effects of CD40-Activated CD40L Reverse Signalling on Inhibitory Medium Spiny Neuron Neurite Growth. Cells, 10(4), 829.

+ Open protocol

+ Expand

Antibody Reagents for Cell Signaling Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols

Rabbit antibodies were as follows; anti-BiP from Sigma or a kind gift of Linda M. Hendershot, anti-phospho-eIF2α (Ser51), anti-phospho-PERK and anti-DARPP-32 (a kind gift of Beth Stevens) from Cell Signaling, anti-GFP from Santa Cruz, anti-CHOP and anti-GADD34 were a kind gift of David Ron. Mouse monoclonal antibodies were as follows; anti-GAPDH from Chemicon International, anti-total eIF2α from Cell Signaling and anti-ATF6 from Abnova. Goat anti-mouse IgG conjugated to Cy2 or to dylight 549, goat anti-rabbit IgG conjugated to Cy3 or to dylight 488, and goat anti-rabbit and anti-mouse IgG conjugated to HRP were from Jackson Labs. Normal goat serum was from Vector Laboratories (Burlingame, CA).

Leitman J., Barak B., Benyair R., Shenkman M., Ashery U., Hartl F.U, & Lederkremer G.Z. (2014). ER Stress-Induced eIF2-alpha Phosphorylation Underlies Sensitivity of Striatal Neurons to Pathogenic Huntingtin. PLoS ONE, 9(3), e90803.

+ Open protocol

+ Expand

Immunohistochemical Analysis of Mouse Brain

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunohistochemical analysis was performed using mouse brains fixed in 4% paraformaldehyde for 12 to 16 hours. Mouse paraffin sections were deparaffinized in xylene followed by rehydration in ethanol diluted in serial dilutions and microwaved in 0.01 M citrate buffer, pH 6.0, at 120 °C for 15 min. The brain area was delimited using a DAKO pen (S2002, Agilent’s Dako, Glostrup, Denmark) and incubated overnight at 4 °C with primary antibodies. The next day, mouse brain sections were washed and incubated with secondary antibodies at room temperature for 1 hour. The antibodies and respective dilutions used included anti-γH2AX antibody, 1:200 (Ser139, #05-636, Millipore, MA, USA); rabbit anti-53BP1, 1:5000 (NB100-304, Novus Biologicals, CO, USA); anti-DARPP-32, 1:200 (Cell Signaling Technology, #2306, MA, USA); anti-Htt, 1:100 (EM48, #MAB5374, Millipore, MA, USA); anti-rabbit IgG Cy3, 1:1000 (711-165-152, Jackson Laboratory, Bar Harbor, ME, USA); anti-mouse IgG Alexa488, 1:1000 (A21202, Molecular Probes, OR, USA); anti-mouse IgG Cy3, 1:1000 (715-165-150, Jackson Laboratory, ME, USA); and anti-rabbit IgG Alexa488, 1:1000 (A21206, Molecular Probes, OR, USA).

Imamura T., Fujita K., Tagawa K., Ikura T., Chen X., Homma H., Tamura T., Mao Y., Taniguchi J.B., Motoki K., Nakabayashi M., Ito N., Yamada K., Tomii K., Okano H., Kaye J., Finkbeiner S, & Okazawa H. (2016). Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries. Scientific Reports, 6, 33861.

+ Open protocol

+ Expand

DARPP-32 Cleavage by Calpain In Vitro

Check if the same lab product or an alternative is used in the 5 most similar protocols

In vitro cleavage of recombinant DARPP-32 WT and T153A proteins by calpain was performed as previously described (Garg et al., 2011 (link)). Briefly, recombinant DARPP-32 WT or T153A (5 μg) was incubated with calpain-1 (Calbiochem) in reaction buffer (50 mm Tris–HCl, pH 7.5, 100 mm NaCl, 3 mm CaCl₂, 2 mm DTT and 1 mm EDTA) for various times (1, 5, 10 and 30 min) with or without calpain inhibitors (100 μm zVAD and 100 μm calpeptin). After being incubated for the indicated times, the reaction mixture was mixed with an equal volume of 2 × SDS sample buffer and boiled for 10 min. Samples were subjected to SDS-PAGE followed by Coomassie staining or Western blotting with anti-DARPP-32 (Cell Signalling, Danvers, MA, USA) or anti-GST (Santa Cruz, Dallas, Texas, USA) antibodies.

Cho K., Cho M.H., Seo J.H., Peak J., Kong K.H., Yoon S.Y, & Kim D.H. (2015). Calpain-mediated cleavage of DARPP-32 in Alzheimer’s disease. Aging Cell, 14(5), 878-886.

+ Open protocol

+ Expand

Western Blot Analysis of Striatal Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Striatal tissue samples were homogenized in a buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (w/v) SDS, 1.0% NP-40, 0.5% sodium deoxycholate, and 1% (v/v) protease inhibitor mixture. For SDS PAGE, 30–50 μg of proteins were separated in a 4–20% gradient gel and transferred to a nitrocellulose membrane. The membrane was blotted with the following primary antibodies: anti-DARPP32 (Cell Signaling, rabbit polyclonal antibody, 1:1000), anti-C1qC (LifeSpan Bioscience, rabbit polyclonal antibody, 1:1000), anti-PSD95 (Thermo Scientific Pierce, mouse monoclonal antibody, 1:1000) and mouse anti-β-actin (Sigma, mouse monoclonal antibody, 1:5000). After incubation with HRP-conjugated secondary antibodies, the bound antibodies were visualized by chemiluminescence. The intensity of the Western blot bands was quantified by Image J software.

Peng Q., Wu B., Jiang M., Jin J., Hou Z., Zheng J., Zhang J, & Duan W. (2016). Characterization of Behavioral, Neuropathological, Brain Metabolic and Key Molecular Changes in zQ175 Knock-In Mouse Model of Huntington’s Disease. PLoS ONE, 11(2), e0148839.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

40 μg of total protein lysate were resolved on SDS-PAGE and immunoblotted with specific antibodies. Anti-phospho-ERK (1:1000) and anti-ERK (1:1000) (all from cell signaling) were used for analysis of the kinase activation. For the analysis of DARPP-32, BDNF expression total lysate were immunoblotted with the anti-DARPP-32 (1:1000; Cell Signaling), anti-BDNF (1:500; Santa Cruz Biotechnology), respectively. Mutant huntingtin aggregates were detected using EM48 antibody (1:1000; Millipore). Anti-aTubulin (1:5000; Abcam, plc, Cambridge, UK) or anti-β-actin (1:3000; Sigma-Aldrich) antibodies were used for protein normalization. HRP-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) were used at 1:5000 dilution. Protein bands were detected by ECL Prime (GE Healthcare) and quantitated with Quantity One (Bio-Rad Laboratories) and/or ImageJ software.

Squitieri F., Di Pardo A., Favellato M., Amico E., Maglione V, & Frati L. (2015). Pridopidine, a dopamine stabilizer, improves motor performance and shows neuroprotective effects in Huntington disease R6/2 mouse model. Journal of Cellular and Molecular Medicine, 19(11), 2540-2548.

+ Open protocol

+ Expand

Immunofluorescent Staining of Brain Slices

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cleared brain slices are placed in PBST. The primary antibody is added at 1:1000 dilution for 2 days, then slices are washed with PBST 5 times for 30 minutes. The secondary antibody is added at 1:1000 overnight in PBST, and the slices washed once for 30 min in PBST. The antibodies used in our experiments were: mouse monoclonal anti-PSD-95 (Pierce, MA1-045), rabbit monoclonal anti-DARPP32 (Cell Signaling 19A3), Alexa Fluor488 Goat anti-rabbit (Life Technologies, A-11008), Alexa Fluor594 Goat anti-mouse (Life Technologies, A-11005).

Poguzhelskaya E., Artamonov D., Bolshakova A., Vlasova O, & Bezprozvanny I. (2014). Simplified method to perform CLARITY imaging. Molecular Neurodegeneration, 9, 19.

+ Open protocol

+ Expand

Immunoblotting of Huntingtin Protein in HD Mouse Models

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tissues collected from zQ175 HD mice were homogenized by dounce homogenizer using cell lysis buffer (Cell Signaling). For R6/2 or Supt4h genetically modified mice, brain lysates were prepared similarly using lysis buffer (10 mM HEPES, 1 mM DTT, 200 μM Na₃VO₄, 8.5% (w/v) sucrose, protease inhibitor). Immunoblotting was performed as previously described [13 (link)]. In brief, equal amounts of protein were resolved by electrophoresis on 8, 12, or 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore), and probed with anti-SUPT4H, anti-α-Tubulin (DM1A, Sigma), anti-TBP (T1827, Sigma), anti-HTT (MAB2166, Chemicon), anti-polyQ (clone 5TF1–1C2, MAB1574, Chemicon), anti-β-actin (GTX109639, GeneTex), anti-GAPDH (GTX100118, GeneTex), or anti-DARPP-32 (#2302, Cell Signaling) antibodies. After incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, the immunoreactive signals were detected by ECL reagent (enhanced chemiluminescence, PerkinElmer).

Cheng H.M., Chern Y., Chen I.H., Liu C.R., Li S.H., Chun S.J., Rigo F., Bennett C.F., Deng N., Feng Y., Lin C.S., Yan Y.T., Cohen S.N, & Cheng T.H. (2015). Effects on Murine Behavior and Lifespan of Selectively Decreasing Expression of Mutant Huntingtin Allele by Supt4h Knockdown. PLoS Genetics, 11(3), e1005043.

+ Open protocol

+ Expand

Immunofluorescent Labelling of DARPP32 and NRG4

Check if the same lab product or an alternative is used in the 5 most similar protocols

Brains were fixed overnight using 4% paraformaldehyde in 0.12 M PBS at 4°C, washed in PBS and cryoprotected in 30% sucrose before being frozen in dry ice-cooled isopentane. Serial 30 μm sections were blocked in 1% bovine serum albumin (Sigma), 0.1% Triton (Sigma) in PBS and then incubated with 1:400 rabbit-monoclonal anti-DARPP32 (Cell Signaling) and 1:200 goat-polyclonal anti-NRG4 (Santa Cruz Biotechnologies, Santa Cruz, CA) antibodies at 4°C overnight. After washing, the sections were incubated with 1:500 of donkey anti-rabbit or goat-polyclonal Alexa-488 or Alexa-647 conjugated secondary antibodies (Invitrogen) for 1 hour at room temperature. Sections were washed, incubated with DAPI, and visualized using a Zeiss LSM710 confocal microscope.

Paramo B., Wyatt S, & Davies A.M. (2019). Neuregulin-4 Is Required for the Growth and Elaboration of Striatal Medium Spiny Neuron Dendrites. Journal of Neuropathology and Experimental Neurology, 78(8), 725-734.

+ Open protocol

+ Expand

Immunocytochemistry of Neuronal Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were fixed for 10 min in 2% paraformaldehyde prepared in PBS. Fixed cells were then washed twice with PBS and permeabilized for 10 min using 0.3% Triton X-100 in PBS. Blocking was performed for 3 hours at 4°C with 5% bovine serum albumin (BSA) in PBS. Next, cells were incubated overnight at 4°C with primary antibodies diluted in PBS with 5% BSA, then washed twice with PBS, and subsequently incubated for 3 hours at room temperature with secondary antibodies diluted in PBS with 5% BSA. After two additional washes with PBS, cells were incubated with Hoechst dye (Invitrogen) diluted 1:10,000 in PBS. The following primary antibodies and dilutions were used: anti-DARPP-32 (1:1000, catalog #2302, Cell Signaling Technology), anti-cFos (1:2000, catalog #sc-52, Lot #C1010, Santa Cruz) and anti-beta tubulin (1:4000, catalog #801213, Lot #B272898, BioLegend) Secondary antibodies were Alexa 488 antimouse (1:1000; A21236) and Alexa 647 anti-rabbit (1:1000; A21245), both from Invitrogen.
Fluorescence imaging of fixed cells was performed in an Opera Phenix ™ high content confocal microscope at 40X magnification.

Jones-Tabah J., Martin R., Tanny J.C., Clarke P.B.S., & Hébert T.E. (2021). Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!