- 44-768G - Invitrogen Antibodies MAPT antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Up-regulation and Antibody-Peptide Competition. Peptide Competition. Extracts of from African green monkey kidney (CV-1) cells were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to nitrocellulose. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with Tau [pSpS199/202] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1), the phosphopeptide immunogen (2), the non-phosphorylated peptide corresponding to the immunogen (3), the tau phosphopeptide corresponding to [pS199] (4), the tau phosphopeptide corresponding to [pS202] (5), or, a generic phosphoserine-containing peptide (6). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase conjugate (Product # ALI4405) and bands were detected using the Tropix WesternStar™ method.The data show that only the phosphopeptide corresponding to Tau [pSpS199/202] completely blocks the antibody signal, demonstrating the specificity of the antibody. (Cat. No. 44768G).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-Tau pSer199/pSer202 Antibody was done on 70% confluent log phase SHSY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-Tau pSer199/pSer202 Antibody (Product # 44-768G) at 1µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 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 Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-Tau (pSpS199/202) showing staining in the cytoplasm of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-Tau (pSpS199/202) polyclonal antibody (Product # 44-768G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Tau [pSpS199/202] was done on SH-SY5Y cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Tau [pSpS199/202] Rabbit Polyclonal Antibody (44768G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 5 Tau phosphorylation is decreased in neurons treated with 20 mM KCl. Immunoblot analysis of Tau phosphorylation in neurons treated either with 10mM or 20mM KCl. The phosphorylation of S199/S202, T181, T205 and S404 was not significantly affected by 10mM of KCl treatment whereas the phosphorylation of all these sites was decreased by 20mM of KCl treatment. The signal of the phospho-antibodies was normalized to that of total Tau (n = 5, mean +- SEM, unpaired t-test two-tailed, * P < 0.05, *** P < 0.001). The amount of dephosphorylated Tau revealed by the Tau-1 antibody was normalized to that of total Tau lysate (n = 5, mean +- SEM, unpaired t-test two-tailed, * P < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Aged mice lacking the WD domain of Atg16L have robust Tau hyperphosphorylation. ( A ) Representative imaging of S199/202 Tau phosphorylation in hippocampus. ( B ) Quantification of Tau phosphorylation in the hippocampus; each point represents and individual mouse. ( C ) CA3 field-specific imaging of S199/202 Tau phosphorylation. ( D ) Immunoblot analysis of whole brain for multiple species of phospho-Tau (pTau) compared to total-Tau. Actin was used as a loading control. *** P < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Female AD/MxD mice have greater Abeta, but not tau, pathology. A Representative image of Abeta plaques. B The number of Abeta plaques/brain section (averaged across 8, 40-muM-thick brain sections between -1.46 and -3.52 mm from bregma). C Hippocampal insoluble Abeta-40, measured by ELISA. D Hippocampal insoluble Abeta-42, measured by ELISA. E Representative Western blot images of tau protein in right (ischemic for MxD) hippocampal isolates. Measurements for F total tau, G pTau S199, and H pTauPHF1 were normalized to GAPDH. Data are presented as mean + SEM, # p < 0.05 effect of sex, ## p < 0.01 effect of sex, 3-way ANOVA, ( n = 4-5/group)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 Immunoblotting using hTg-Tau and Tau-KO brain lysates confirms the specificity for tau for a panel of antibodies. a ) Scheme of tau (2N4R) showing the epitopes of the Tau12, Tau5, Tau1, HT7, BT2 and Dako antibodies. b ) - d' ) Immunoblots demonstrate strong reactivity of all antibodies with tau bands between 40 and 60 kD. Minor non-specific bands present in Tau-KO brain lysates were detected in d ), e ), i ), h ), p ), x ), y ) and c '). Isoform-specific antibodies in h ), i ), j ) and k ) were validated using a tau ladder with all 6 recombinant human tau isoforms. kDa sizes for the marker are given in e' ). Tg: hTg-Tau mouse, KO: Tau-KO mouse brain lysate, TL: recombinant tau ladder

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Compared to Braak 0-I samples, many but not all, tau phosphorylation events are increased in native Braak III-IV samples. Normalized phospho-tau signals obtained from ELISA measurements of samples from a ) entorhinal cortices (EC), b ) hippocampi (Hip) and c ) temporal cortices (TC). Student's t-tests: *, p < 0.05, **, p < 0.01, ***, p < 0.001

44-768G

Antibody data