Overview

  • Product name
    Anti-Histone H1.2 antibody - ChIP Grade
    See all Histone H1.2 primary antibodies
  • Description
    Rabbit polyclonal to Histone H1.2 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    This antibody has only been tested on bulk HeLa histones. We expect this antibody to be specific for Histone H1.2 - however we have not tested this specifically on different histone H1 variants.
  • Tested applications
    Suitable for: ChIP, WB, IP, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide derived from residues 1 - 100 of Human Histone H1.2.

    Read Abcam's proprietary immunogen policy .

  • Positive control
    • HeLa Histone preparation
  • General notes

    Histone H1.2 is one of five known h1 variants (known as H1.1/2/3/4/5 and/or H1.a/b/c/d/e). The H1 variants differ from each other in the amino acid sequence of their N-terminal regions.

Properties

Applications

Our Abpromise guarantee covers the use of ab4086 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 Use 2-6 µg for 25 µg of chromatin.
WB 1/500. Detects a band of approximately 25 kDa (predicted molecular weight: 21 kDa).
IP Use a concentration of 5 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function
    Histones H1 are necessary for the condensation of nucleosome chains into higher order structures.
  • Sequence similarities
    Belongs to the histone H1/H5 family.
    Contains 1 H15 (linker histone H1/H5 globular) domain.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H1 histone family member 2 antibody
    • H1.a antibody
    • H12_HUMAN antibody
    • H1F2 antibody
    • H1s-1 antibody
    • HIST1H1C antibody
    • Histone 1 H1c antibody
    • Histone cluster 1 H1c antibody
    • Histone H1.2 antibody
    • Histone H1c antibody
    • Histone H1d antibody
    • Histone H1s-1 antibody
    • MGC3992 antibody
    see all

Images

  • ab4086 Histone H1.2

    The antibody was used at a dilution of 1/500

    Lane 1: HeLa Histone (5ug) + ab4086 
    Lane 2: HeLa Histone (5ug) + ab4086 + 1µg/ml of peptide (Histone H1.2) (ab16936)

    Secondary ab: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

    Exposure time: 1 minute
    Expected molecular weight: 21.3 kDa

    ab4086 Histone H1.2

    The antibody was used at a dilution of 1/500

    Lane 1: HeLa Histone (5ug) + ab4086
    Lane 2: HeLa Histone (5ug) + ab4086 + 1µg/ml of peptide (Histone H1.2) (ab16936)

    Secondary ab: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

    Exposure time: 1 minute
    Expected molecular weight: 21.3 kDa
  • Histone H1.2 - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab4086.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 21kDa; Histone H1.2 - ChIP Grade

  • ICC/IF image of ab4086 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 10 min) and incubated with the antibody (ab4086, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab4086 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
        

  • IHC image of Histone H1.2 staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab4086, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

References

This product has been referenced in:
  • Chiarella E  et al. ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells. Stem Cell Rev N/A:N/A (2018). Read more (PubMed: 29938352) »
  • Liao R & Mizzen CA Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation. Epigenetics Chromatin 10:29 (2017). Read more (PubMed: 28539972) »
See all 14 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
Western blot
Sample
Spermophilus tridecemlineatus Tissue lysate - whole (Liver)
Gel Running Conditions
Reduced Denaturing (12.5%)
Loading amount
15 µg
Specification
Liver
Blocking step
BSA as blocking agent for 7 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C

Mr. James Bjork

Verified customer

Submitted Aug 24 2015

Answer

Thank you for contacting us.

Your credit note ID is xxx.

I am sorry that these 2 antibodies did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department.

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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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 xxx for1 vial of ab4086 with a new lot. As for ab5212, order xxx has been arranged with 1x vial oflot 863574. Theestimated delivery date for this vial is May 25th. I apologize about this delay.

Please let me know how each lot is working out for you and I will send you the second vial for each antibody.

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, and look forward to hear back from you.

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Question
Answer

Thank you very much for your telephone enquiry.

I am sorry to confirm that to our knowledge, the two histone H1.2 antibodies we have are regrettably not tested and guaranteed in mouse or frozen sections (IHC-Fr).

ab17677 Anti-Histone H1.2 antibody
Rabbit polyclonal
ICC/IF, IHC-P, IP, WB
Reacts with:Human

ab4086 Anti-Histone H1.2 antibody - ChIP Grade
Rabbit polyclonal
ChIP, ICC/IF, IHC-P, WB
Reacts with: Human

I have checked the alignment of the immunogen for these antibodies with the mouse Histone H1.2 protein. This is very quite low at 63%. Unfortunately, this means I would suggest there is likely to be little crossreactivity of these antibodies with the mouse histone H1.2 protein.

Therefore, unfortunately it is not possible for use to offer any testing discount on this occasion.

If I am sorry we have no suitably tested Histone H1.2 antibodies for your requirements on this occasion. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for contacting us. We are pleased to hear that you attended our seminar on the 16th and hope you enjoyed it. I appreciate the time you have spent in the laboratory and understand your concerns. We would like to investigate this particular case further for you and offer some suggestions to help optimise the results for your patient tissue samples. Have you tried to keep the samples on ice during the sonication procedure? For such a long sonication it is recommended to cool the samples throughout the cycles, repeatedly adding fresh ice if necessary. Also, do you cross-link your samples? The crosslinking in ChIP serves to create a snapshot of the cell at the moment when you add the formaldehyde, as it kills cells immediately and crosslinks protein to DNA. This step also has influence on the sonication efficiency. For details please find our protocol attached. May I ask if this sonication step has been optimised? Ideally, DNA fragments should be approximately 300 bp in length and it is advisable to perform a time course to determine the optimal sonication conditions. So depending on the sample type a shorter procedure may be suitable as well. Although some ChIP grade antibodies may recognize the native structure of the antigen, I would not recommend proceeding to the next step without cross-linking and sonicating the samples. I hope this information is helpful and would like to wish you good luck with your research. The technical team is always at your service, should you require further expert advice. PS: I am happy to let you know that we have a webinar on ChIP coming up (presumably in January) that might be of interest to you. Details can be found in due course on our website (https://www.abcam.com/index.html?pageconfig=tradeshow).

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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 (MRC5VA)
Loading amount
15 µg
Specification
MRC5VA
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Sep 01 2009

Answer

Thank you for your enquiry. This antibody was tested on purified core histones purchased from another company. They were purified by acid extraction precipitation from log phase untreated HeLa cells. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for your e-mail. We are very pleased to hear that you are satisfied with the antibody ab4086. I enclose our recommended protocol for blocking, please do not hesitate to contact us again if you need further information. In principle, a small volume of antibody (e.g., 1-5 ul) is first reacted with excess peptide (5-50 fold over the antibody; e.g., 1 ug antibody reacted with 5-50 ug peptide; exact amounts determined by titration) to neutralize it. The neutralized antibody can no longer subsequently bind to another antigen (a band of interest) or staining pattern. So the band(s)/staining that is competed by the antigen/peptide is supposedly specific. If more than one band disappears by peptide/antigen competition then those bands have the antigenic determinants and could be considered either fragments of the large antigen or multimer. 1. Determine the amount of antibody that is needed for 1 strip (e.g., 2 ml). For example, an antibody has given desired bands at 1:1000 dilution. So you will need 1 ul/ml antibody (2-ul antibody for 2-ml antibody solution). If an antibody were used at 1:5000 dilution then you would only need 0.2 ul/ml (use 2 ul of 1:10 dilution for better accuracy). 2. Take 2-ul antibody (or as needed) in 100 ul saline/PBS. Make 2 tubes. Add antigen/peptide solution (10-50 ug peptide or antigen added in 10-100 ul). Add same volume of saline/PBS (no peptide/antigen) to the other tube labeled as -peptide or No peptide. Mix gently. 3. Incubate both tubes at 37oC for 1-2 hrs or 2-24 hrs at 4oC. 4. Centrifuge the tubes for 15 min at 4oC in a microfuge (10-15000 rpm) to pellet any immune complexes. Carefully remove the supernatant. If no visible pellet is seen than just leave approx. 5-10 ul at the bottom to avoid disturbing invisible immune complexes. If you do not centrifuge the solution, it may give high background. 5. After centrifugation, make up the volume of supt. to 2 ml (or what is necessary depending upon the initial antibody taken) with buffer (PBS-Tween with or without BSA or milk or any buffer that was used initially). Use both antibodies (with and without antigen/peptide) for Western blotting or immunolocalization. 4. Observe the bands/staining that disappears. We have tested ab4086 specificity by mixing it at 1/500 with 1µg/ml of peptide Histone H1.2 ab16936.

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