Anti-TATA binding protein TBP antibody [1TBP18] - ChIP Grade (ab818)

Mouse monoclonal TATA binding protein TBP antibody [1TBP18]. Validated in WB, IP, ELISA, IHC, ICC, EMSA, Flow Cyt, ChIP, ChIP/Chip, ICC/IF and tested in Mouse, Rat, Human. Cited in 186 publication(s).

Overview

  • Product name

    Anti-TATA binding protein TBP antibody [1TBP18] - ChIP Grade
    See all TATA binding protein TBP primary antibodies
  • Description

    Mouse monoclonal [1TBP18] to TATA binding protein TBP - ChIP Grade
  • Host species

    Mouse
  • Tested applications

    Suitable for: ChIP/Chip, ELISA, IHC-P, EMSA, IP, WB, Flow Cyt, ChIP, ICC/IF, ICC, IHC-Frmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Does not react with: Saccharomyces cerevisiae, Xenopus laevis, Drosophila melanogaster, Silk worm
  • Immunogen

    Synthetic peptide (Human).

  • Epitope

    Within amino acid residues 1-20 of human, mouse and rat TBP. The specific epitope for this antibody is accessible when the C-terminal domain of TBP is removed or when TBP is complexed with DNA.
  • Positive control

    • HeLa nuclear extract
  • General notes

    ab81216 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

    This product was changed from ascites to tissue culture supernatant on 19/12/2018. Please note that the dilutions may need to be adjusted accordingly. If you have any questions please do not hesitate to contact our scientific support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab818 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
ChIP/Chip Use at an assay dependent concentration.
ELISA 1/1000.

We recommend Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) (ab6729) secondary antibody.

IHC-P Use at an assay dependent concentration.
EMSA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/2000. Detects a band of approximately 38 kDa (predicted molecular weight: 38 kDa). PubMed: 23767827
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody

ChIP Use 5-10 µg for 25 µg of chromatin.
ICC/IF Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

Target

  • Function

    General transcription factor that functions at the core of the DNA-binding multiprotein factor TFIID. Binding of TFIID to the TATA box is the initial transcriptional step of the pre-initiation complex (PIC), playing a role in the activation of eukaryotic genes transcribed by RNA polymerase II. Component of the transcription factor SL1/TIF-IB complex, which is involved in the assembly of the PIC (preinitiation complex) during RNA polymerase I-dependent transcription. The rate of PIC formation probably is primarily dependent on the rate of association of SL1 with the rDNA promoter. SL1 is involved in stabilization of nucleolar transcription factor 1/UBTF on rDNA.
  • Tissue specificity

    Widely expressed, with levels highest in the testis and ovary.
  • Involvement in disease

    Defects in TBP are the cause of spinocerebellar ataxia type 17 (SCA17) [MIM:607136]. Spinocerebellar ataxia is a clinically and genetically heterogeneous group of cerebellar disorders. Patients show progressive incoordination of gait and often poor coordination of hands, speech and eye movements, due to degeneration of the cerebellum with variable involvement of the brainstem and spinal cord. SCA17 is an autosomal dominant cerebellar ataxia (ADCA) characterized by widespread cerebral and cerebellar atrophy, dementia and extrapyramidal signs. The molecular defect in SCA17 is the expansion of a CAG repeat in the coding region of TBP. Longer expansions result in earlier onset and more severe clinical manifestations of the disease.
  • Sequence similarities

    Belongs to the TBP family.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Database links

  • Alternative names

    • GTF2D antibody
    • GTF2D1 antibody
    • HDL4 antibody
    • MGC117320 antibody
    • MGC126054 antibody
    • MGC126055 antibody
    • SCA17 antibody
    • TATA binding factor antibody
    • TATA box factor antibody
    • TATA sequence binding protein antibody
    • TATA sequence-binding protein antibody
    • TATA-binding factor antibody
    • TATA-box binding protein N-terminal domain antibody
    • TATA-box factor antibody
    • TATA-box-binding protein antibody
    • TBP antibody
    • TBP_HUMAN antibody
    • TFIID antibody
    • Transcription initiation factor TFIID TBP subunit antibody
    see all

Images

  • All lanes : Anti-TATA binding protein TBP antibody [1TBP18] - ChIP Grade (ab818) at 1/2000 dilution

    Lane 1 : Human Huh7 nuclear cell lysate
    Lane 2 : Human Huh7 cytoplast cell lysate
    Lane 3 : Human HepG2 nuclear cell lysate
    Lane 4 : Human HepG2 cytoplast cell lysate

    Lysates/proteins at 40 µg per lane.

    Secondary
    All lanes : HRP-conjugated goat polyclonal to mouse IgG at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 38 kDa
    Observed band size: 38 kDa


    Exposure time: 30 seconds


    This image was generated using the ascites version of the product. 

    See Abreview

  • Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 8 µg of  ab818 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The last wash was performed with final wash buffer containing 250 mM NaCl. The immunoprecipitated DNA was quantified by real time PCR (Taqman and sybr green approach). Primers and probes are located in the core promoter region of the genes.

    This image was generated using the ascites version of the product. 

  • ab818 staining TATA binding protein TBP in Human stomach tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The tissue underwent formaldehyde fixation before heat mediated antigen retrieval in 10mM Citrate buffer pH 6.0. Blocking was done in 5% serum for 1 hour at 23°C. The primary antibody was diluted 1/100 and incubated with the sample for 1 hour at 23°C. An HRP-conjugated Goat polyclonal to Mouse IgG was used undiluted as the secondary antibody.

    This image was generated using the ascites version of the product. 

     

    See Abreview

  • Overlay histogram showing HeLA cells stained with ab818 (red line). The cells were fixed with 100% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab818, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was Mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    This image was generated using the ascites version of the product.

  • ab818 staining TATA binding protein TBP in NIH3T3 mouse fibroblast cells by Immunocytochemistry/ Immunofluorescence. The cells were fixed in formaldehyde, permeabilised in 0.025% Triton X and then blocked using 5% serum for 1 hour at 23°C. Samples were then incubated with primary antibody at 1/200 for 16 hours at 4°C. The secondary antibody used was a goat anti-mouse IgG conjugated to Alexa Fluor® 350 (blue) used at a 1/1000 dilution.

    This image was generated using the ascites version of the product. 

    See Abreview

  • ab818 staining TATA binding protein TBP by ELISA. The sample was purified from human AGS gastric carcinoma cell line and blocked with 5% BSA for 1 hour at 25°C. The primary antibody was used at 1/1000 dilution, and incubated with sample for 16 hour at 4°C. Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) (ab6729) secondary antibody was used as secondary, diluted at 1/1000.

    This image was generated using the ascites version of the product. 

    See Abreview

  • Anti-TATA binding protein TBP antibody [1TBP18] - ChIP Grade (ab818) at 1/2000 dilution + Ros C cells with endogenous TBP

    Performed under reducing conditions.

    Predicted band size: 38 kDa



    Review by Jilin Liu submitted 19 August 2004.

    This image was generated using the ascites version of the product. 

References

This product has been referenced in:

  • Xu W  et al. Plant-derived alkaloid sinomenine potentiates glucocorticoid pharmacodynamics in mitogen-activated human peripheral blood mononuclear cells by regulating the translocation of glucocorticoid receptor. Phytother Res 33:187-196 (2019). Read more (PubMed: 30357956) »
  • Lee SH  et al. 18F-FDG positron emission tomography as a novel diagnostic tool for peripheral nerve injury. J Neurosci Methods 317:11-19 (2019). Read more (PubMed: 30684510) »
See all 195 Publications for this product

Customer reviews and Q&As

1-10 of 32 Q&A

Answer

You can see this antibody used in a gel shift assay in Thompson NE et al. 2004 Protein Expression and Purification 36: 186-197. However, this publication (which must be purchased to view the entire article) refers to using a protocol for the gel shift assay found in Wiley SR et al. 1991 Proc Natl Acad Sci USA 89: 5814-5818 (available free of charge) with the following modifications:

A 25bp oligonucleotide containing a consensus TATA element was purchased from Promega. The reaction volumes were 20ul and contained 8ng of 5’-end-labeled 32P-labeled oligonucleotide and 10ng of human TATA Binding Protein. The reactions were incubated at 23oC for 15 minutes. For the MAb supershift assays, 40ng of MAb was added, and the reactions were allowed to incubate at 23oC for an additional 15 minutes. The reactions were subjected to electrophoresis in a nondenaturing gradient (4-15%) polyacrylamide gel on a PhastSystem for a total of 140-300Vh. The gel and buffer strips were first soaked in a solution of 1/2x TBE and 2mM MgCl2; the MgCl2 helped to stabilize the TBP/DNA complex in the absence of TFIIB.

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Answer

Thank you for completing our survey.

I am sorry to hear the replacement product also didn't work as expected. I would like to offer you a credit as this product is still not working as expected. Please could you confirm whether this is to your satisfaction.

I look forward to your reply.

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Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.
I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement of ab1791 with the order number 1161045.
To check the status of the order please contact our Customer Service team and reference this number.
Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.
I wish you the best of luck with your research.

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Answer

Thank you for your updates. I am very sorry to hear that even after following the suggestions, the antibody still does not perform as it is expected.
I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
We have alternative antibodies against the same target TBP (ab51841) or against Laminin B1 (ab16048). Please could you take a look at the datasheets of these products and let me how you wish to proceed with your enquiry.
I look forward to hearing from you soon.

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Answer

Thank you for contacting us.

We are happy to help customers find the most suitable products for their research purposes . In our review of this problem it does seem that cisplatin may affect the levels of TBP in cells while nucleolin seems affected mainly in it's distribution; however we are not specialized in this particular field and do not want to recommend a product that may be unsuitable. We encourage customers to consult the latest literature available through PubMed and other resources in order to find the most up-to-date information about their specific research interests.


I am sorry that I could not be more helpful, but I hope that the available literature in this area can provide some clarification. Please do not hesitate to contact us again with other needs or with any questions about our products.

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Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 123456.
To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question

1) Abcam product code ab818

2) Abcam order reference number or product batch number
Lot GR59271-4

3) Description of the problem

No band detected on western blot.

4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): nuclear extracts.
Lysis buffer: 0.01M Tris-Cl pH 7.4, 0.01M NaCl, 0.003M MgCl2, 0.5% triton X-100
Lysis buffer: 40% glycerol, 0.05M Tris-Cl pH 7.4, 0.005M MgCl2, 0.1mM EDTA
Sample: HEK293T
Protease inhibitors: complete EDTA free (Roche)
Phosphatase inhibitors: No
Reducing agent beta-mercaptoethanol
Boiling for ≥5 min? YES 10min 99°C
Protein loaded ug/lane or cells/lane not quite sure…..
Positive control NO
Negative control NO

5) Percentage of gel 12%
Type of membrane PVDF
Protein transfer verified YES
Blocking agent and concentration 5%Milkpowder in PBST
Blocking time 1hour
Blocking temperature RT

6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1/500-1/2000
Diluent buffer 5%Milkpowder in PBST
Incubation time 2h RT or overnight 4°C
Incubation temperature:

7) Secondary antibody: anti mouse IgG HRP
Species: goat
Reacts against: mouse
Concentration or dilution 1/2000
Diluent buffer 5%Milkpowder in PBST
Incubation time 1h
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP

8) Washing after primary and secondary antibodies:
Buffer PBST
Number of washes 4 times 10min or 3 times 5min

9) Detection method: ECL

10) How many times have you run this staining? Five or six times with different settings
Do you obtain the same results every time? YES
What steps have you altered to try and optimize the use of this antibody?

Antibod dilution
Antibody incubation time
Blocking buffer dilution
Wash steps reduced

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

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Answer

Thank you for your enquiry regarding ab818 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) It would be worth trying RIPA buffer for preparing the samples to prevent any unwanted protein degradation during the procedure. The composition of this buffer can be found at this site:
https://www.abcam.com/index.html?pageconfig=resource&rid=11379
2) Try to load at least 20 or 40 ug protein per lane. If it is not possible to quantify the protein concentration in the samples, increase the loading material 2x or 4x to see if the signal is getting stronger or not.
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

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Answer

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.
In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.
As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.
Please could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

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Question


>1) Abcam product code ab818
>
>2) Abcam order reference number or product batch number: GR59271-2, Do
>you have an order number or PO number?
I can't find the order number.
>3) Description of the problem: Band at 60 kDa instead of 38 kDa. Are
>there any faint bands at the 60 kDa?
They are very distinct bands. I attached the scan. The right 3lanes are
nuclear fraction. Huge band at 38kDa in left 3lanes are leftover of GAPDH.
>
>4) Sample preparation:
>Type of sample (whole cell lysates, fraction, recombinant protein…):
>human peripheral eosinophils
>Lysis buffer: NE-PER kit (Pierce)
>Protease inhibitors: Halt Protease/Phosphatase inhibitor cocktail (Thermo)
>Phosphatase inhibitors: ↑
>Reducing agent: Pierce 5x sample buffer with DTT
>Boiling for ≥5 min? No (3min)
> Protein loaded ug/lane or cells/lane: aprox. 4µg (200µg/mL x 20µL)
>Positive control: none
>Negative control: noen
>
>5) Percentage of gel: 4˜12% gradient
>Type of membrane: Nitrocellulose
>Protein transfer verified: n/a
>Blocking agent and concentration: 5%BSA
>Blocking time: 1hr
>Blocking temperature: RT
>
>6) Primary antibody (If more than one was used, describe in “additional
>notes”) :
>Concentration or dilution: 1000:1
> Diluent buffer: TBS-t w/1%BSA
>Incubation time: O/N
>Incubation temperature: 4˚C
>
>7) Secondary antibody:
>Species: Goat
>Reacts against: Rabbit
>Concentration or dilution: 2500:1
>Diluent buffer: TBS-t w/1%BSA
>Incubation time: 1hr
>Incubation temperature: RT
>Fluorochrome or enzyme conjugate: none
>
>8) Washing after primary and secondary antibodies:
>Buffer: TBS-t
>Number of washes: 4
>
>9)Detection method: Chemiluminescence (Luminata Forte)
>10) How many times have you run this staining?: 2
>Do you obtain the same results every time?: y
>What steps have you altered to try and optimize the use of this
>antibody?: Different dilution
>
>Document attachment: Attaching images of your blot is strongly
>recommended and can greatly speed up our investigation of your problem.

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Do you know about when you ordered this product? I see an order from July 2009. Is this your order?
You may want to try boiling the samples longer in case there are any mulitmers forming. Try boiling 5-10 mins.
You had mentioned that you're using a goat anti-rabbit secondary, but the primary is raised in a mouse. Was this a mistake? Plus it was mentioned that the secondary wasn't conjugated to anything but that chemiluminescent detection was used. Was the secondary actually HRP-conjugated?
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.

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Question
Answer

Thank you for contacting us.

Your credit note ID is ***.

I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

The credit note ID is for your reference only and does not automatically guarantee the credit.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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1-10 of 32 Q&A

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