Anti-GAL4 antibody (ab1396)
Key features and details
- Rabbit polyclonal to GAL4
- Suitable for: WB, ChIP
- Reacts with: Saccharomyces cerevisiae
- Isotype: IgG
Overview
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Product name
Anti-GAL4 antibody
See all GAL4 primary antibodies -
Description
Rabbit polyclonal to GAL4 -
Host species
Rabbit -
Specificity
Customers feedbacks suggests that this antibody would not provide satisfactory results in Drosophila melanogaster. -
Tested applications
Suitable for: WB, ChIPmore details -
Species reactivity
Reacts with: Saccharomyces cerevisiae
Does not react with: Drosophila melanogaster -
Immunogen
Synthetic peptide corresponding to Saccharomyces cerevisiae GAL4 aa 100-200 conjugated to keyhole limpet haemocyanin.
(Peptide available asab23612) -
Positive control
- This antibody gave a positive signal with GAL4-VP16 recombinant protein.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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ChIP Related Products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab1396 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application Abreviews Notes WB Use a concentration of 1 µg/ml. Predicted molecular weight: 99 kDa.ChIP Use at an assay dependent concentration.Notes WB
Use a concentration of 1 µg/ml. Predicted molecular weight: 99 kDa.ChIP
Use at an assay dependent concentration.Target
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Relevance
Function: This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which encode for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. Subunit structure: Binds DNA as a homodimer. Interacts directly with the mediator subunits GAL11/MED15 and SRB4/MED17. Domain: The 9aaTAD motif (residues 862 to 870) is a transactivation domain present in a large number of yeast and animal transcription factors. Post-translational modification: Association between GAL11 and GAL4 may serve to expedite phosphorylation of GAL4. -
Cellular localization
Nuclear -
Database links
- Entrez Gene: 855828 Saccharomyces cerevisiae
- SwissProt: P04386 Saccharomyces cerevisiae
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Alternative names
- Gal4p antibody
- GAL81 antibody
- Regulatory protein GAL4 antibody
Images
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Western blot - Anti-GAL4 antibody (ab1396)
Anti-GAL4 (ab1396) antibody at 1ug/ml.
Lane 1: Marker.
Lane 2A: Whole cell yeast lysate control from untransformed SEY6210 pep4-3 strain (a kind gift from Prof. Tom Stevens from University of Oregon).
Lane 2B: Whole cell yeast lysate from SEY6210 pep4-3 strain transformed with pJK22-pGBDU plasmid containing GAL4 DNA Binding domain fused to the VPS60 Gene (a kind gift from Prof. Tom Stevens from University of Oregon).
Lane 2C: Whole cell yeast lysate from SEY6210 pep4-3 strain transformed with pJk23-pGAD plasmid containing GAL4 Active Domain fused to the VPS60 Gene (a kind gift from Prof. Tom Stevens from University of Oregon).
Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1/10000 dilution.
Predicted band size: 99 kDa
Additional bands: 56 kDa (possible isoform, Fusion Protein)
Developed using the ECL technique. Performed under reducing conditions with exposure time of 5 mins. The samples run on a 4-20% gradient gel. All blocking and antibody incubation steps done in 5% milk in 20mM Tris-HCl plus 0.1% TWEEN-20.
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ChIP - Anti-GAL4 antibody (ab1396)This image is courtesy of the Tony Kouzarides lab at the Gurdon Institute.A stably transfected 293T human cell line harbouring the GAL4 upstream activation sequence was transiently transfected with a V5 or T7- tagged GAL4 DNA Binding Domain construct. 48 hours post transfection Chromatin was prepared according to the Abcam X-ChIP protocol. The ChIP was performed with 25 ug chromatin, 5ug of antibody and 20 ul of Protein A/G beads. A non-specific antibody was used as the negative control. The immunoprecipitated DNA was quantified by real time PCR (SYBR Green approach). -
ChIP - Anti-GAL4 antibody (ab1396)This image is courtesy of an anonymous collaborator.S. cerevisiae cells were incubated in raffinose-containing media then transferred to galactose-containing media to activate transcription of galactose activated genes. IP was performed with 500µl of cell extract incubated overnight with 5µl of ab1396 at 4°C, followed by the addition of 25µl of protein A sepharose beads and incubation for 2 hours (room temp). The subsequently purified DNA was analysed with 3 pairs of primers (see diagram). The results show that Gal4 binds specifically to the UAS region of Gal1-Gal10 in both raffinose and galactose, despite only 500-600bp between the UAS and 5' primer.
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Western blot - Anti-GAL4 antibody (ab1396)Anti-GAL4 antibody (ab1396) at 1 µg/ml + GAL4-VP16 Recombinant Protein at 0.1 µg
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 99 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?
Exposure time: 2 minutes
ab1396 was tested against GAL4-VP16 Recombinant Protein predicted to run at 28 kDa.
Datasheets and documents
References (10)
ab1396 has been referenced in 10 publications.
- Elison GL et al. Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter. Cell Rep 25:737-748.e4 (2018). PubMed: 30332652
- Li J et al. Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification. Sci Rep 6:35631 (2016). WB . PubMed: 27762338
- Roedgaard M et al. DNA Topoisomerases Are Required for Preinitiation Complex Assembly during GAL Gene Activation. PLoS One 10:e0132739 (2015). WB, ChIP ; Saccharomyces cerevisiae . PubMed: 26173127
- Yang J et al. GEMIN4 functions as a coregulator of the mineralocorticoid receptor. J Mol Endocrinol 54:149-60 (2015). PubMed: 25555524
- Chen X et al. Activation of a human chromosomal replication origin by protein tethering. Nucleic Acids Res 41:6460-74 (2013). WB . PubMed: 23658226
- Inturi R et al. Adenovirus precursor pVII protein stability is regulated by its propeptide sequence. PLoS One 8:e80617 (2013). WB . PubMed: 24260437
- Dawson MA et al. Three distinct patterns of histone H3Y41 phosphorylation mark active genes. Cell Rep 2:470-7 (2012). WB . PubMed: 22999934
- Knutson BA & Hahn S Domains of Tra1 important for activator recruitment and transcription coactivator functions of SAGA and NuA4 complexes. Mol Cell Biol 31:818-31 (2011). ChIP ; Saccharomyces cerevisiae . PubMed: 21149579
- Pinskaya M et al. H3 lysine 4 di- and tri-methylation deposited by cryptic transcription attenuates promoter activation. EMBO J 28:1697-707 (2009). ChIP ; Saccharomyces cerevisiae . PubMed: 19407817
- Ghosh M et al. Transcription factor binding and induced transcription alter chromosomal c-myc replicator activity. Mol Cell Biol 24:10193-207 (2004). WB . PubMed: 15542830
Images
-
Western blot - Anti-GAL4 antibody (ab1396)
Anti-GAL4 (ab1396) antibody at 1ug/ml.
Lane 1: Marker.
Lane 2A: Whole cell yeast lysate control from untransformed SEY6210 pep4-3 strain (a kind gift from Prof. Tom Stevens from University of Oregon).
Lane 2B: Whole cell yeast lysate from SEY6210 pep4-3 strain transformed with pJK22-pGBDU plasmid containing GAL4 DNA Binding domain fused to the VPS60 Gene (a kind gift from Prof. Tom Stevens from University of Oregon).
Lane 2C: Whole cell yeast lysate from SEY6210 pep4-3 strain transformed with pJk23-pGAD plasmid containing GAL4 Active Domain fused to the VPS60 Gene (a kind gift from Prof. Tom Stevens from University of Oregon).
Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1/10000 dilution.
Predicted band size: 99 kDa
Additional bands: 56 kDa (possible isoform, Fusion Protein)
Developed using the ECL technique. Performed under reducing conditions with exposure time of 5 mins. The samples run on a 4-20% gradient gel. All blocking and antibody incubation steps done in 5% milk in 20mM Tris-HCl plus 0.1% TWEEN-20.
-
ChIP - Anti-GAL4 antibody (ab1396) This image is courtesy of the Tony Kouzarides lab at the Gurdon Institute.A stably transfected 293T human cell line harbouring the GAL4 upstream activation sequence was transiently transfected with a V5 or T7- tagged GAL4 DNA Binding Domain construct. 48 hours post transfection Chromatin was prepared according to the Abcam X-ChIP protocol. The ChIP was performed with 25 ug chromatin, 5ug of antibody and 20 ul of Protein A/G beads. A non-specific antibody was used as the negative control. The immunoprecipitated DNA was quantified by real time PCR (SYBR Green approach).
-
ChIP - Anti-GAL4 antibody (ab1396) This image is courtesy of an anonymous collaborator.
S. cerevisiae cells were incubated in raffinose-containing media then transferred to galactose-containing media to activate transcription of galactose activated genes. IP was performed with 500µl of cell extract incubated overnight with 5µl of ab1396 at 4°C, followed by the addition of 25µl of protein A sepharose beads and incubation for 2 hours (room temp). The subsequently purified DNA was analysed with 3 pairs of primers (see diagram). The results show that Gal4 binds specifically to the UAS region of Gal1-Gal10 in both raffinose and galactose, despite only 500-600bp between the UAS and 5' primer.
-
Western blot - Anti-GAL4 antibody (ab1396)Anti-GAL4 antibody (ab1396) at 1 µg/ml + GAL4-VP16 Recombinant Protein at 0.1 µg
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 99 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?
Exposure time: 2 minutes
ab1396 was tested against GAL4-VP16 Recombinant Protein predicted to run at 28 kDa.
