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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [6]
- Immunocytochemistry [2]
- Immunohistochemistry [2]
- Chromatin Immunoprecipitation [1]
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- Product number
- PA5-27649 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- TRIM28 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: A431, H1299, HeLa, HepG2, NIH-3T3, mouse brain. Predicted reactivity: Mouse (91%), Rat (91%), Bovine (94%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 0.31 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using TRIM28 Polyclonal Antibody (Product # PA5-27649). Sample (30 µg of whole cell lysate). A: Hela . B: Hep G2 . 7.5% SDS PAGE. TRIM28 Polyclonal Antibody (Product # PA5-27649) diluted at 1:10,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using TRIM28 Polyclonal Antibody (Product # PA5-27649). Sample (30 µg of whole cell lysate). A: NIH-3T3. 7.5% SDS PAGE. TRIM28 Polyclonal Antibody (Product # PA5-27649) diluted at 1:1,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using TRIM28 Polyclonal Antibody (Product # PA5-27649). Sample (50 µg of whole cell lysate). Lane A: Mouse brain . 7.5% SDS PAGE. TRIM28 Polyclonal Antibody (Product # PA5-27649) diluted at 1:1,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockout of TRIM28 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR801077_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of TRIM28 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa TRIM28 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein 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 Anti-TRIM28 Polyclonal Antibody (Product # PA5-27649, 1:5,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to TRIM28.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of TRIM28 was achieved by transfecting HEK-293 with TRIM28 specific siRNAs (Silencer® select Product # s19778, s19780). Western blot analysis (Fig. a) was performed using Whole cell extracts from the TRIM28 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TRIM28 Polyclonal Antibody (Product # PA5-27649, 1:5000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 TRIM28.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-TRIM28 Polyclonal Antibody(Product # PA5-27649) and a 100kDa band corresponding to TRIM28 was observed along with uncharacterized bands (*) across cell lines and tissues tested. Whole cell extracts (1% SDS) (30 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2), Hep G2 (Lane 3), HT-1080 (Lane 4) and NIH/3T3 (Lane 5).Tissue extracts of Mouse Liver (Lane 6) and Mouse Thymus (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). Expression of TRIM28 was observed to be high in Mouse Thymus in comparison to low or negative in Mouse Liver. The blot was probed with the primary antibody (1:5000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection 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 KAP1 in paraformaldehyde-fixed HeLa cells using a KAP1 polyclonal antibody (Product # PA5-27649) at a 1:200 dilution.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of TRIM28 was performed using 70% confluent log phase 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 45 minutes at room temperature. The cells were labeled with TRIM28 Polyclonal Antibody (Product # PA5-27649) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), 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 dilution). Panel d represents the merged image showing Nuclear 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
- TRIM28 Polyclonal Antibody detects KAP1 protein at nucleus on mouse ovary by immunohistochemical analysis. Sample: Paraffin-embedded mouse ovary. TRIM28 Polyclonal Antibody (Product # PA5-27649) diluted at 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- TRIM28 Polyclonal Antibody detects KAP1 protein at nucleus on mouse ovary by immunohistochemical analysis. Sample: Paraffin-embedded mouse ovary. TRIM28 Polyclonal Antibody (Product # PA5-27649) diluted at 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Chromatin Immunoprecipitation (ChIP) assay of endogenous TRIM28 protein using TRIM28 Antibody: ChIP was performed using TRIM28 Polyclonal Antibody (Product # PA5-27649, 5 µg) on sheared chromatin from HeLa cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CDC25- Promoter, RBL1-Promoter, CCNE2- Promoter and CCBL1 (active) and SAT alpha satellite repeats and Actin beta (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
