Overview

  • Product name
  • Description
    Rabbit polyclonal to SATB2
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, EMSA, IHC-FoFr, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 700 to the C-terminus of Mouse SATB2.

    Read Abcam's proprietary immunogen policy (Peptide available as ab34906.)

  • Positive control
    • Brain (Mouse) Whole Cell Lysate - normal tissue, 0 days old.

Properties

Applications

Our Abpromise guarantee covers the use of ab34735 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. Detects a band of approximately 85 kDa (predicted molecular weight: 83 kDa).
EMSA Use at an assay dependent concentration. PubMed: 22123820
IHC-FoFr Use at an assay dependent concentration. PubMed: 19709629
ICC/IF Use at an assay dependent concentration. PubMed: 19232401

Target

  • Function
    Binds to DNA, at nuclear matrix- or scaffold-associated regions. Thought to recognize the sugar-phosphate structure of double-stranded DNA. Transcription factor controlling nuclear gene expression, by binding to matrix attachment regions (MARs) of DNA and inducing a local chromatin-loop remodeling. Acts as a docking site for several chromatin remodeling enzymes and also by recruiting corepressors (HDACs) or coactivators (HATs) directly to promoters and enhancers. Required for the initiation of the upper-layer neurons (UL1) specific genetic program and for the inactivation of deep-layer neurons (DL) and UL2 specific genes, probably by modulating BCL11B expression. Repressor of Ctip2 and regulatory determinant of corticocortical connections in the developing cerebral cortex. May play an important role in palate formation. Acts as a molecular node in a transcriptional network regulating skeletal development and osteoblast differentiation.
  • Tissue specificity
    High expression in adult brain, moderate expression in fetal brain, and weak expression in adult liver, kidney, and spinal cord and in select brain regions, including amygdala, corpus callosum, caudate nucleus, and hippocampus.
  • Involvement in disease
    Note=Chromosomal aberrations involving SATB2 are found in isolated cleft palate. Translocation t(2;7); translocation t(2;11).
    Defects in SATB2 are a cause of cleft palate isolated (CPI) [MIM:119540]. A congenital fissure of the soft and/or hard palate, due to faulty fusion. Isolated cleft palate is not associated with cleft lips. Some patients may manifest other craniofacial dysmorphic features, mental retardation, and osteoporosis.
    Note=A chromosomal aberration involving SATB2 is found in a patient with classical features of Toriello-Carey syndrome. Translocation t(2;14)(q33;q22).
  • Sequence similarities
    Belongs to the CUT homeobox family.
    Contains 2 CUT DNA-binding domains.
    Contains 1 homeobox DNA-binding domain.
  • Post-translational
    modifications
    Sumoylated by PIAS1. Sumoylation promotes nuclear localization, but represses transcription factor activity.
  • Cellular localization
    Nucleus matrix.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA binding protein SATB2 antibody
    • DNA-binding protein SATB2 antibody
    • FLJ21474 antibody
    • FLJ32076 antibody
    • GLSS antibody
    • KIAA1034 antibody
    • MGC119474 antibody
    • MGC119477 antibody
    • SATB family member 2 antibody
    • SATB homeobox 2 antibody
    • SATB2 antibody
    • SATB2_HUMAN antibody
    • Special AT rich sequence binding protein 2 antibody
    • Special AT-rich sequence-binding protein 2 antibody
    see all

Images

  • Anti-SATB2 antibody (ab34735) at 1 µg/ml + Mouse brain tissue lysate - total protein (0 days) (ab7188) at 10 µg

    Secondary
    IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

    Performed under reducing conditions.

    Predicted band size: 83 kDa
    Observed band size: 85 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 30 kDa. We are unsure as to the identity of these extra bands.

  • ab34735 used in EMSA.
    EMSA demonstrating binding of SATB1 and SATB2 to an oligonucleotide containing a predicted SATB-binding element located 125 bp upstream of the Eomes transcription start site (-125). Labeled SATB probes were incubated with Rcho-1 TS cell nuclear lysates as indicated above the lanes. Lane 1, without any competitor oligonucleotide; lane 2, with unlabeled wild-type competitor; lane 3, with unlabeled mutant competitor; lane 4, with SATB1 antibody; lane 5, with SATB2 antibody (ab34735); lane 6, with a control antibody. B and C, Rcho-1 TS cells stably transfected with pcDNA3, pcDNA3-HA-Satb1, or pcDNA3-HA-Satb2 and maintained in stem culture conditions were used for ChIP assays. Antibodies to the HA tag (Anti-HA) or acetylated histone H3K9 (Anti-AcH3) were used in the analyses. IgG was used as a control for nonspecific immunoprecipitation. qPCR analyses were performed on immunoprecipitates using primers amplifying the segment (-172 to -63) including the SATB-bin
  • ab34735 staining SATB2 in human osteosarcoma SAOS-2 cells by Immunocytochemistry/ Immunofluorescence.
    Cells were fixed in paraformaldehyde, permeabilized using PBS/ 0.25% Triton, blocked with 1% BSA for 1 hour at room temperature and then incubated with ab34735 at a 1/500 dilution for 1 hour. The secondary used was an Alexa-Fluor 488 conjugated goat anti-rabbit polyclonal used at a 1/250 dilution.

    See Abreview

References

This product has been referenced in:
  • Che A  et al. Layer I Interneurons Sharpen Sensory Maps during Neonatal Development. Neuron 99:98-116.e7 (2018). Read more (PubMed: 29937280) »
  • Chu P  et al. The Impact of Perineuronal Net Digestion Using Chondroitinase ABC on the Intrinsic Physiology of Cortical Neurons. Neuroscience 388:23-35 (2018). Read more (PubMed: 30004010) »
See all 24 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (human osteosarcoma SAOS-2 cell line)
Specification
human osteosarcoma SAOS-2 cell line
Fixative
Paraformaldehyde
Permeabilization
Yes - PBS-TRITON 0.25%
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Apr 19 2012

Answer

Thank you for providing the details of your protocol.

Have you tried using the primary antibody at a 1:1000 dilution? Because this is a polyclonal antibody, there may be some batch to batch variability. Our internal QC will only pass this antibody if it gives good signal at the dilution stated on our datasheet (1:1000). While the lot you had previously purchased may have been a particularly strong batch and worked at 1:2500, I would recommend using more primary antibody if you are not seeing any signal.

If you would like to try a new batch, I will be happy to send you one as a free of charge replacement. Unfortunately, I was unable to locate a record of your order in our system. Did you by any chance purchase this antibody through our Canadian distributor Cedarlane? If so, can you please tell me the approximate day of purchase so that I can locate the correct order? Once I have found your order I will be happy to send you a free of charge replacement from a new lot.

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Answer

Thank you for contacting us. I am sorry to hear that this batch is not working for your western blot. It is certainly possible that there may be a batch specific issue with the lot you received. If that is the case and you have purchased the antibody in the past six months, I will be happy to send you a free of charge replacement with a new lot, or offer a credit or refund.

It would be very helpful for our complaints investigation if you could please provide some details of your protocol and results. Were you getting high background, no signal, or an incorrect molecular weight? It would be very helpful if you could send an image of your blot. What dilutions and incubation times were used with the primary and secondary antibodies? What was the blocking agent and how were the samples prepared? Was thisa reduced / denatured blot? What was your original order number?

I will be happy to assist you further once I have this additional information.

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Question
Answer

DISCOUNT CODE: ******* Expiration date: ******** I am very pleased to hear you would like to accept our offer and test ab34735 on human samples. This code will give you one free primary antibody before the expiration date. To redeem this offer, please submit a western blot Abreview for human samples and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. We publish positive and negative Abreviews on our datasheets so please submit the results of your tests regardless of the result. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (NT2D1 cells)
Loading amount
10 µg
Specification
NT2D1 cells
Gel Running Conditions
Reduced Denaturing (10% gel, SDS buffer used for running in BioRad Protean tetracell)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 03 2012

Answer

As we just discussed on the phone, to process you free of charge replacement, I do not confirmation from your collaborator that it is ok for me to send the new antibody to you. Once I receive the confirmation, I will have the new antibody you requested, ab51502 sent to you.

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Answer

Looking the images you sent, the blot on the bottom right, shows a band at approximately 75kDa and a band below 100kDa, could this be the 85kDa band? There is also a band just above that. Does SATB2 undergo any post-translational modifications that could account for the higher band? As I mentioned previously, this antibody is covered under our Abpromise. Therefore as this antibody is not working as guaranteed, then I would be happy to send you another SABT2 antibody as a replacement.

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Question

Here's a brief summary of what I did so far: 1st attempt: no signal at all (1:1000) 2nd attempt, at Tom's recommendation tried 1% BSA, and the whole membrane was black (on the film) (1:500) 3rd attempt, described below, with photos attached From left to right in all the photos: Ladder-Ctrl1-Ctrl2-Treated1-Treated2-empty-empty-Treated2-Treated1-Ctrl2-Ctrl1-Ladder The left half was blocked with milk, the right half was blocked with BSA (see below) each lane contains 120ug of whole cell lysate extracted from human immortalized BEAS-2B cells (bronchial epithelial cells) ran on an "anyKD" TGX pre-cast gel from Bio-Rad per Bio-Rad recommendations used a Bio-Rad protein kaleidoscope on each end of the gel (size marked on film - pink band is 75kDa, this protein should around 85kDa) transferred to a PVDF membrane, cut the membrane in half, and blocked half with 5% milk and half with 3% BSA for one hour (both in TBST) used SATB2 antibody (ab 34735) at 1:500 overnight at 4oC (diluted Ab in milk for half, BSA for half) I snipped on a little piece of the corner of each half and did not use any primary Ab on these pieces as a control. rinsed with TBST for >2 hours total (3 rinses) used secondary goat anti-rabbit at 1:2000 (from Santa Cruz - I have used this numerous times with no problems) rinsed for 30 minutes total in TBST (3 rinses) added ECL reagent and developed for 1 minute (see film) I used a lot of protein since I used only 50ug before (when I saw nothing) - I wanted to ensure that lack of protein was not the problem. The mRNA for this protein is highly upregulated (>20x) in the treated cells and there is no literature to suggest that it is regulated at the level of mRNA, so I would have expected the band to be strongest in lanes 4,5,8, and 9. I see nothing in the milk-blocked half and 6-7 sets of bands in the BSA-blocked half. The membrane photo is inverted in color, so those strange black lines look more like tiny white bubbles on the actual membrane, but they don't seem to correlate at all to the bands on the film/gel, so I think they are unrelated to the problem. I'd appreciate your help, thanks.

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Answer

As the images are black and white, I am unable to determine which band is the 75kDa pink band you mention in your methods. Could you send me a labeled version of the blot, so that I can see where the band of interest should be. In general though, you do seem to be getting a lot of background, no matter if you use 5% milk or 3% BSA as a blocking reagent. I can only think of a few things that may be causing such high background and the presence of multiple lanes. Firstly, since you almost trebled the amount of protein loaded and then doubled the primary antibody concentration, that combination may have caused such a high noisy signal. I understand you loaded more protein, to make sure that the lack of protein was not an issue, but in case there may be too much protein was loaded. So I would suggest either reducing the amount of protein loaded and keeping the primary antibody concentration the same or vice versa. As for the multiple bands, how fresh is your lysis buffer? Also what type of protease inhibitors are you using? When you see the ladder effect in your lanes, it could be that you either need to use fresh lysis buffer or increase the concentration of protease inhibitors in your buffer. I would just like to say that under our Abpromise, we do guarantee all of our antibodies for 6 months to work as stated on the datasheet. In this case, ab34735 is guaranteed to work in western blot and on human samples. So if we are not able to able to get this antibody working successfully for you, then we will try and find another that will. I hope some of what i have said helps and please let me know if there is anything else I can do

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