49-1036

antibody from Invitrogen Antibodies

Targeting: TBP GTF2D1, SCA17, TFIID

Antibody data

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Validation data

TBP protein structure - 49-1036 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of TBP was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR639612_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of TBP was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) andHeLa TBP KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with TBP Monoclonal Antibody (Product # 49-1036, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5000 dilution) using the iBright™ FL 1500 (Product # A44115). 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 TBP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Whole cell extracts (40 μg) from HeLa cells transfected with TBP siRNA (lane 2) and from an untransfected control (lane 1) were analysed by Western blot using the anti-TBP antibody (Product # 49-1036) diluted 1:500 in TBSTween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TBP was achieved by transfecting HeLa with TBP specific siRNAs (Silencer® select Product # s13826, s13827). Western blot analysis (Fig. a) was performed using nuclear enriched extracts from the TBP knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2), and untransfected cells (lane 1). The blot was probed with TBP Monoclonal Antibody (Product # 49-1036, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knockdown confirms that antibody is specific to TBP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-TBP Monoclonal Antibody (Product # 49-1036) and a 41kDa band corresponding to TBP was observed across all cell lines tested except for HT-29 where 37 kDa band was also observed. Nuclear enriched extracts (30 µg lysate) of K-562 (Lane 1), HeLa (Lane 2), A-431 (Lane 3), HT-29 (Lane 4) and NIH/3T3 (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (1:500 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: ChIP assays were performed using human osteosarcoma (U-2 OS) cells, the anti-TBP antibody (Product # 49-1036), optimized PCR primer sets, myoglobin exon 2, GAPDH promoter, GAPDH promoter (0.5 kb), and GAPDH promoter (1.0 kb) for qPCR. Each ChIP experiment used sheared chromatin from 1 million cells and 4 µg of anti-TBP antibody. Recovery (% ChIP⁄input) is shown in the top graph. Occupancy (fold: +ve⁄-ve) is in the bottom graph. Recovery and occupancy of the c-fos promoter by TBP (red bars); recovery and occupancy of the GAPDH promoter by TBP (green bars) are shown. Occupancy of the two promoters by TBP is evident based on fluorescent qPCR analysis of immunoprecipitated DNA. Controls for IP and PCR specificity include primers for the myoglobin exon 2 (black) as well as GAPDH (0.5 kb, pink) and GAPDH (1 kb, blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP was performed with 5 µg of the anti-TBP antibody (Product # 49-1036) on sheared chromatin from 1 million HeLaS3 cells. The IP’d DNA was analysed by QPCR with optimized PCR primer pairs for the promoters of the active GAPDH and c-fos genes, used as positive control targets, and for a region 1 kb upstream of the GAPDH promoter and the coding region of the inactive MB gene, used as negative control targets (figure A). The IP’d DNA was subsequently analysed with an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the peak distribution in 50 kb regions surrounding the GAPDH, c-fos, ACTB and MCL1 genes (figure B, C, D and E, respectively). These results clearly show a localisation of TBP at the promoters of actively transcribed genes.