PA1-741

antibody from Invitrogen Antibodies

Targeting: DLG1 dJ1061C18.1.1, DLGH1, hdlg, SAP-97, SAP97

Provider product page for PA1-741

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [30]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [6]
Other assay [22]

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Product number: PA1-741 - Provider product page
Provider: Invitrogen Antibodies
Product name: SAP97 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-741 detects Synapse-Associated Protein 97 (SAP97) from human, rat and mouse tissues. No cross-reactivity to other proteins, including synapse-associated protein family members, has been observed. PA1-741 has been successfully used in Western blot, immunofluorescence, immunocytochemistry, immunoprecipitation, and immunohistochemistry procedures. By Western blot, this antibody detects an ~140 kDa protein representing SAP97 from rat brain extract. Immunohistochemical staining of SAP97 in rat hippocampus with PA1-741 results in intense neuronal cell body and dendrite staining. The PA1-741 immunizing peptide corresponds to amino acids 115-133 from rat SAP97. This sequence is 84% and 74% conserved in mouse and human SAP97, respectively. PA1-741 can be used with blocking peptide PEP-055.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

DLG1 protein structure - PA1-741 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of SAP97 was performed by loading 25 µg of C6 (lane 1), PC12 (lane 2) and rat brain (lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a SAP97 polyclonal antibody (Product # PA1-741) at a dilution of 1:5000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~140 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SAP97 Polyclonal Antibody (Product # PA1-741) and a 103 kDa band corresponding to DLG1 was observed in neural stem cells (NSC) differentiated into neurons and Mouse Cerebellum along with an uncharacterized band (*) was NSC. Whole cell extracts (40 µg lysate) of NSC (Lane 1), NSC differentiated into neurons (Lane 2), and tissue extract (40 µg lysate) of Mouse Cerebellum (Lane 3) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescentdetection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of SAP97 (green) showing staining in the cytoplasm and membrane of C2C12 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a SAP97 polyclonal antibody (Product # PA1-741) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of SAP97 (green) showing staining in the cytoplasm and membrane of HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a SAP97 polyclonal antibody (Product # PA1-741) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of SAP97 (green) showing staining in the cytoplasm and membrane of NIH-3T3 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a SAP97 polyclonal antibody (Product # PA1-741) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SAP97 was performed using 70% confluent log phase RSC96 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with SAP97 Rabbit Polyclonal Antibody (Product # PA1-741) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of DLG1 was performed using neural stem cells (NSC) differentiated into neurons. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with SAP97 Polyclonal Antibody (Product # PA1-741) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing plasma membrane and synaptic junctional localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of DLG1 was performed using neural stem cells (NSC) differentiated into neurons. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with SAP97 Polyclonal Antibody (Product # PA1-741) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing plasma membrane and synaptic junctional localization. Panel e represents low level expression in undifferentiated NSCs. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 7 CRHR1-WT and CRHR1-STAVA localize throughout the neuronal plasma membrane including the excitatory post synapse. Primary hippocampal neurons at DIV 14-20 were transduced with CRHR1-WT and CRHR1-STAVA using AAV8. (A) The spatial pattern of CRHR1-WT and CRHR1-STAVA expression visualized by immunostaining of the GFP tag was confirmed using an anti-CRHR1 antibody. (B) Microtubule-associated protein 2 (MAP2) staining revealed the dendritic presence of the receptor. (C) Co-staining with the axon initial segment marker ankyrin G demonstrated CRHR1-WT and CRHR1-STAVA localization within axons. (D) The presynaptic marker synapsin indicated that CRHR1-WT and CRHR1-STAVA are present in the adjacent post synapse but not in the presynaptic axon terminal. (E) CRHR1-WT and CRHR1-STAVA did not co-localize with the inhibitory postsynaptic marker gephyrin but (F) they co-localized with the MAGUK and excitatory postsynaptic marker PSD95 in spines. (G) CRHR1-WT and mutant co-localized with the candidate interaction partner SAP97.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 8 CRHR1 PDZ binding motif is required for clustering with interacting MAGUKs. CRHR1-WT (A), CRHR1-STAVA (G), or the interacting proteins (first row) were transiently transfected alone. (B-F) Wild-type CRHR1 or (H-L) CRHR1-STAVA were transiently transfected in HEK293 together with (B, H) PSD95-flag, (C, I) HA-SAP97, (D, J) flag-SAP102, (E, K) PSD93-GFP and (F, L) myc-MAGI2. CRHR1-WT co-transfection with the respective interacting partner resulted in a co-clustering of both proteins (B-F). (H-L) Co-transfection of CRHR1-STAVA with the respective interacting partner did not result in any clustering, but both proteins appeared with a subcellular distribution similar to the individual transfections. Immunostaining was performed against the HA tag of HA-CRHR1 or HA-CRHR1-STAVA when co-transfected with PSD95-flag or flag-SAP102. Immunostaining was performed against the flag tag of flag-CRHR1 or flag-CRHR1-STAVA when co-transfected with PSD93-GFP, HA-SAP97, or myc-MAGI2 respectively.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 SAP97 protein is sufficiently knocked down in SAP97-cKO animals. (A) Western blots showing reduced SAP97 band intensity in cerebellum, hippocampus, and cortex. (B) Quantification of western blot analysis. *P < .05, **P < .01 (two-tailed Student's t test). Data are presented as mean +- SEM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 SAP97 expression in human HD and PD hippocampus and striatum. (a) Significant increases in SAP97 expression in the post-mortem human hippocampus in HD and PD patients. The significant increase in SAP97 expression occurs in all hippocampal regions examined: DG, area CA3, and area CA1. * P < 0.05, ** P < 0.005. (b) No significant changes in SAP97 expression were observed in HD or PD striatum in either the caudate nucleus (CN) or the putamen (Put). (c) Representative images of SAP97 immunostaining in the dentate gyrus and CA3 and CA1 hippocampal regions. (d) Representative striatal images of SAP97 immunostaining in the putamen and caudate nucleus. Scale bar 25 mu m for (c) and (d). (e) High power example image of SAP97 immunolabeling, showing the expected somatic and dendritic localisations.

Supportive validation

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Experimental details: Figure 5 Quantitative immunohistochemistry of SAP97, PSD-95, GluN1, and GluA2 expression in YAC128 hippocampal sections. Sections were prepared from symptomatic 1-year-old YAC128 mice to provide a comparison to the end stage of human HD. (a)-(d). Top: Quantification of (a) SAP97, (b) PSD-95, (c) GluA2, and (d) GluN1 levels in dentate gyrus (DG), area CA3, and area CA1. Below: Example immunohistochemical staining for each glutamatergic synaptic protein in the hippocampal CA1 region in control (wildtype) and YAC128 mice. (e) Immunohistochemical quantification of DARPP-32 expression in wild-type and YAC128 striatum (caudate putamen, CPu). * P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 Validation of the identified proteins in dendritic filopodia. (A,B) Localization of the identified proteins in dendritic filopodia (A1-A21) and phagocytic cups (B1-B21) at 14 DIV hippocampal neurons. The cultured neurons were immunostained with anti-TLCN antibody and specific antibodies against Galphao (A1,B1) , Na + /K + ATPase alpha3 (A2,B2) , EFA6D (A3,B3 ), spectrin (A4,B4) , septin7 (A5,B5) , myosin VA (A6,B6) , Galphaq (A7,B7) , Gbeta2 (A8,B8) , eEF1gamma (A9,B9) , ribosomal protein S16 (A10,B10) , CaMKIIalpha (A11,B11) , CD98hc (A12,B12) , EFA6C (A13,B13) , BAIAP2L1 (A14,B14) , PLCbeta3 (A15,B15) , MRCKalpha (A16,B16) , NR3A/B (GluN3A/B) (A17,B17) , SAP97 (SLC3A2) (A18,B18) , MAP1S (A19,B19) , EPS8L1 (A20,B20) , and JIP-4 (SPAG9) (A21,B21) . In merged images, the identified proteins and TLCN are shown in magenta and green, respectively. Single plane of images focused on dendritic filopodia (A1-A21) and center of microbeads (B1-B21) were acquired using a confocal microscopy. Scale bars, 5 mum in (A14,A21,B14,B21) .