Antibody data
- Antibody Data
- Antigen structure
- References [1]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Other assay [1]
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Validation data
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- Product number
- PA5-18822 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- 14-3-3 theta Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with canine, mouse and rat based on sequence homology.
- Reactivity
- Human
- Host
- Goat
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
Submitted references Prospective evaluation of the lymph node proteome in dogs with multicentric lymphoma supplemented with sulforaphane.
Parachini-Winter C, Bracha S, Ramsey SA, Yang L, Ho E, Leeper HJ, Curran KM
Journal of veterinary internal medicine 2020 Sep;34(5):2036-2047
Journal of veterinary internal medicine 2020 Sep;34(5):2036-2047
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot staining of Human Hippocampus lysate using Product # PA5-18822 at a concentration of 0.03 µg/mL, the primary antibody incubation was 1 hour and the detection method was chemiluminescence.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using anti-14-3-3 theta Polyclonal Antibody (Product # PA5-18822) and a 28kDa band corresponding to 14-3-3 theta was observed across cell lines tested. Whole cell extracts (1%SDS) (30 µg lysate) of OVCAR-3 (Lane 1), SH-SY5Y (Lane 2) and HeLa (Lane 3) were electrophoresed using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). 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 dilution) and detected by chemiluminescence with Rabbit anti-Goat IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of 14-3-3 theta was achieved by transfecting HeLa with 14-3-3 theta siRNAs (Silencer® select Product # s21599). Western blot analysis (Fig. a) was performed using whole cell extracts from the 14-3-3 theta knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with 14-3-3 theta recombinant polyclonal antibody (Product # PA5-18822, 1:1000 dilution) and Rabbit anti-Goat IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to 14-3-3 theta.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of 14-3-3 theta was performed using HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with 14-3-3 theta Goat Polyclonal Antibody (Product # PA5-18822) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Product # A-11078) at a dilution of 1:2000 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 composite image showing cytoplasmic localization of 14-3-3 theta in HeLa cells . 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
- FIGURE 2 Immunoblots of lymph node samples for 7 dogs (d) with naive multicentric lymphoma before (D0) and after (D7) supplementation with sulforaphane. The proteins HIP, A, and 14-3-3-theta, B, were detected in all samples (top rows). The immunoblots for beta-actin (second rows) were used to normalize the band volume of HIP and 14-3-3-theta. The normalized expression ratio at D7/D0 is the ratio of the normalized band volume at D7 vs D0 for each dog (third rows). For comparison, the corresponding fold change at D7 vs D0 previously determined by LC-MS/MS is also annotated (bottom rows). FC D7/D0: Fold change at D7 vs D0 (mass spectrometry); NA: Not applicable; NER D7/D0: Normalized expression ratio at D7 vs D0 (immunoblot)