Overview

  • Product name
    Anti-HMGB1 antibody - ChIP Grade
    See all HMGB1 primary antibodies
  • Description
    Rabbit polyclonal to HMGB1 - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-FoFr, ICC/IF, IHC-P, ChIP, IHC-Fr, ICC, ELISA, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Rabbit, Cow
  • Immunogen

    Synthetic peptide within Human HMGB1 aa 150 to the C-terminus (internal sequence) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
    Database link: P09429
    (Peptide available as ab18650)

  • Positive control
    • Recombinant Human HMGB1 protein (ab73658) can be used as a positive control in WB. This antibody gave a positive signal in the following whole cell lysates:HeLa, Jurkat, A431, HEK 293, NIH 3T3, MEF1, PC12. This antibody gave a positive signal in the following nuclear lysate: HeLa nuclear lysate
  • General notes

      

Properties

Applications

Our Abpromise guarantee covers the use of ab18256 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
IHC-FoFr Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use at an assay dependent concentration.
IHC-Fr 1/100.
ICC Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 29 kDa (predicted molecular weight: 25 kDa).

Target

  • Function
    Multifunctional redox sensitive protein with various roles in different cellular compartments. In the nucleus is one of the major chromatin-associated non-histone proteins and acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V(D)J recombination, DNA repair and genome stability. Proposed to be an universal biosensor for nucleic acids. Promotes host inflammatory response to sterile and infectious signals and is involved in the coordination and integration of innate and adaptive immune responses. In the cytoplasm functions as sensor and/or chaperone for immunogenic nucleic acids implicating the activation of TLR9-mediated immune responses, and mediates autophagy. Acts as danger associated molecular pattern (DAMP) molecule that amplifies immune responses during tissue injury. Released to the extracellular environment can bind DNA, nucleosomes, IL-1 beta, CXCL12, AGER isoform 2/sRAGE, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and activates cells through engagement of multiple surface receptors. In the extracellular compartment fully reduced HMGB1 (released by necrosis) acts as a chemokine, disulfide HMGB1 (actively secreted) as a cytokine, and sulfonyl HMGB1 (released from apoptotic cells) promotes immunological tolerance (PubMed:23519706, PubMed:23446148, PubMed:23994764, PubMed:25048472). Has proangiogdenic activity (By similarity). May be involved in platelet activation (By similarity). Binds to phosphatidylserine and phosphatidylethanolamide (By similarity). Bound to RAGE mediates signaling for neuronal outgrowth (By similarity). May play a role in accumulation of expanded polyglutamine (polyQ) proteins such as huntingtin (HTT) or TBP (PubMed:23303669, PubMed:25549101).
    Nuclear functions are attributed to fully reduced HGMB1. Associates with chromatin and binds DNA with a preference to non-canonical DNA structures such as single-stranded DNA, DNA-containing cruciforms or bent structures, supercoiled DNA and ZDNA. Can bent DNA and enhance DNA flexibility by looping thus providing a mechanism to promote activities on various gene promoters by enhancing transcription factor binding and/or bringing distant regulatory sequences into close proximity (PubMed:20123072). May have an enhancing role in nucleotide excision repair (NER) (By similarity). However, effects in NER using in vitro systems have been reported conflictingly (PubMed:19446504, PubMed:19360789). May be involved in mismatch repair (MMR) and base excision repair (BER) pathways (PubMed:15014079, PubMed:16143102, PubMed:17803946). May be involved in double strand break repair such as non-homologous end joining (NHEJ) (By similarity). Involved in V(D)J recombination by acting as a cofactor of the RAG complex: acts by stimulating cleavage and RAG protein binding at the 23 bp spacer of conserved recombination signal sequences (RSS) (By similarity). In vitro can displace histone H1 from highly bent DNA (By similarity). Can restructure the canonical nucleosome leading to relaxation of structural constraints for transcription factor-binding (By similarity). Enhances binding of sterol regulatory element-binding proteins (SREBPs) such as SREBF1 to their cognate DNA sequences and increases their transcriptional activities (By similarity). Facilitates binding of TP53 to DNA (PubMed:23063560). Proposed to be involved in mitochondrial quality control and autophagy in a transcription-dependent fashion implicating HSPB1; however, this function has been questioned (By similarity). Can modulate the activity of the telomerase complex and may be involved in telomere maintenance.
    In the cytoplasm proposed to dissociate the BECN1:BCL2 complex via competitive interaction with BECN1 leading to autophagy activation (PubMed:20819940). Involved in oxidative stress-mediated autophagy (PubMed:21395369). Can protect BECN1 and ATG5 from calpain-mediated cleavage and thus proposed to control their proautophagic and proapoptotic functions and to regulate the extent and severity of inflammation-associated cellular injury (By similarity). In myeloid cells has a protective role against endotoxemia and bacterial infection by promoting autophagy (By similarity). Involved in endosomal translocation and activation of TLR9 in response to CpG-DNA in macrophages.
    In the extracellular compartment (following either active secretion or passive release) involved in regulation of the inflammatory response. Fully reduced HGMB1 (which subsequently gets oxidized after release) in association with CXCL12 mediates the recruitment of inflammatory cells during the initial phase of tissue injury; the CXCL12:HMGB1 complex triggers CXCR4 homodimerization (PubMed:22370717). Induces the migration of monocyte-derived immature dendritic cells and seems to regulate adhesive and migratory functions of neutrophils implicating AGER/RAGE and ITGAM (By similarity). Can bind to various types of DNA and RNA including microbial unmethylated CpG-DNA to enhance the innate immune response to nucleic acids. Proposed to act in promiscuous DNA/RNA sensing which cooperates with subsequent discriminative sensing by specific pattern recognition receptors (By similarity). Promotes extracellular DNA-induced AIM2 inflammasome activation implicating AGER/RAGE (PubMed:24971542). Disulfide HMGB1 binds to transmembrane receptors, such as AGER/RAGE, TLR2, TLR4 and probably TREM1, thus activating their signal transduction pathways. Mediates the release of cytokines/chemokines such as TNF, IL-1, IL-6, IL-8, CCL2, CCL3, CCL4 and CXCL10 (PubMed:12765338, PubMed:18354232, PubMed:19264983, PubMed:20547845, PubMed:24474694). Promotes secretion of interferon-gamma by macrophage-stimulated natural killer (NK) cells in concert with other cytokines like IL-2 or IL-12 (PubMed:15607795). TLR4 is proposed to be the primary receptor promoting macrophage activation and signaling through TLR4 seems to implicate LY96/MD-2 (PubMed:20547845). In bacterial LPS- or LTA-mediated inflammatory responses binds to the endotoxins and transfers them to CD14 for signaling to the respective TLR4:LY96 and TLR2 complexes (PubMed:18354232, PubMed:21660935, PubMed:25660311). Contributes to tumor proliferation by association with ACER/RAGE (By similarity). Can bind to IL1-beta and signals through the IL1R1:IL1RAP receptor complex (PubMed:18250463). Binding to class A CpG activates cytokine production in plasmacytoid dendritic cells implicating TLR9, MYD88 and AGER/RAGE and can activate autoreactive B cells. Via HMGB1-containing chromatin immune complexes may also promote B cell responses to endogenous TLR9 ligands through a B-cell receptor (BCR)-dependent and ACER/RAGE-independent mechanism (By similarity). Inhibits phagocytosis of apoptotic cells by macrophages; the function is dependent on poly-ADP-ribosylation and involves binding to phosphatidylserine on the cell surface of apoptotic cells (By similarity). In adaptive immunity may be involved in enhancing immunity through activation of effector T cells and suppression of regulatory T (TReg) cells (PubMed:15944249, PubMed:22473704). In contrast, without implicating effector or regulatory T-cells, required for tumor infiltration and activation of T-cells expressing the lymphotoxin LTA:LTB heterotrimer thus promoting tumor malignant progression (By similarity). Also reported to limit proliferation of T-cells (By similarity). Released HMGB1:nucleosome complexes formed during apoptosis can signal through TLR2 to induce cytokine production (PubMed:19064698). Involved in induction of immunological tolerance by apoptotic cells; its pro-inflammatory activities when released by apoptotic cells are neutralized by reactive oxygen species (ROS)-dependent oxidation specifically on Cys-106 (PubMed:18631454). During macrophage activation by activated lymphocyte-derived self apoptotic DNA (ALD-DNA) promotes recruitment of ALD-DNA to endosomes.
  • Tissue specificity
    Ubiquituous. Expressed in platelets (PubMed:11154118).
  • Sequence similarities
    Belongs to the HMGB family.
    Contains 2 HMG box DNA-binding domains.
  • Domain
    HMG box 2 mediates proinflammatory cytokine-stimulating activity and binding to TLR4 (PubMed:12765338, PubMed:20547845). However, not involved in mediating immunogenic activity in the context of apoptosis-induced immune tolerance (PubMed:24474694).
    The acidic C-terminal domain forms a flexible structure which can reversibly interact intramolecularily with the HMG boxes and modulate binding to DNA and other proteins (PubMed:23063560).
  • Post-translational
    modifications
    Phosphorylated at serine residues. Phosphorylation in both NLS regions is required for cytoplasmic translocation followed by secretion (PubMed:17114460).
    Acetylated on multiple sites upon stimulation with LPS (PubMed:22801494). Acetylation on lysine residues in the nuclear localization signals (NLS 1 and NLS 2) leads to cytoplasmic localization and subsequent secretion (By similarity). Acetylation on Lys-3 results in preferential binding to DNA ends and impairs DNA bending activity.
    Reduction/oxidation of cysteine residues Cys-23, Cys-45 and Cys-106 and a possible intramolecular disulfide bond involving Cys-23 and Cys-45 give rise to different redox forms with specific functional activities in various cellular compartments: 1- fully reduced HMGB1 (HMGB1C23hC45hC106h), 2- disulfide HMGB1 (HMGB1C23-C45C106h) and 3- sulfonyl HMGB1 (HMGB1C23soC45soC106so).
    Poly-ADP-ribosylated by PARP1 when secreted following stimulation with LPS.
    In vitro cleavage by CASP1 is liberating a HMG box 1-containing peptide which may mediate immunogenic activity; the peptide antagonizes apoptosis-induced immune tolerance (PubMed:24474694). Can be proteolytically cleaved by a thrombin:thrombomodulin complex; reduces binding to heparin and proinflammatory activities.
  • Cellular localization
    Nucleus. Chromosome. Cytoplasm. Secreted. Cell membrane. Endosome. Endoplasmic reticulum-Golgi intermediate compartment. In basal state predominantly nuclear. Shuttles between the cytoplasm and the nucleus (PubMed:12231511, PubMed:17114460). Translocates from the nucleus to the cytoplasm upon autophagy stimulation (PubMed:20819940). Release from macrophages in the extracellular milieu requires the activation of NLRC4 or NLRP3 inflammasomes (By similarity). Passively released to the extracellular milieu from necrotic cells by diffusion, involving the fully reduced HGMB1 which subsequently gets oxidized (PubMed:19811284). Also released from apoptic cells (PubMed:16855214, PubMed:18631454). Active secretion from a variety of immune and non-immune cells such as macrophages, monocytes, neutrophils, dendritic cells and natural killer cells in response to various stimuli such as LPS and cytokines involves a nonconventional secretory process via secretory lysosomes (PubMed:12231511, PubMed:14532127, PubMed:15944249). Secreted by plasma cells in response to LPS (By similarity). Found on the surface of activated platelets (PubMed:11154118).
  • Information by UniProt
  • Database links
  • Alternative names
    • Amphoterin antibody
    • Chromosomal protein, nonhistone, HMG1 antibody
    • DKFZp686A04236 antibody
    • High mobility group 1 antibody
    • High mobility group box 1 antibody
    • High mobility group protein 1 antibody
    • High mobility group protein B1 antibody
    • high-mobility group (nonhistone chromosomal) protein 1 antibody
    • HMG-1 antibody
    • HMG1 antibody
    • HMG3 antibody
    • HMGB 1 antibody
    • HMGB1 antibody
    • HMGB1_HUMAN antibody
    • NONHISTONE CHROMOSOMAL PROTEIN HMG1 antibody
    • SBP 1 antibody
    • Sulfoglucuronyl carbohydrate binding protein antibody
    see all

Images

  • All lanes : Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1 µg/ml

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : HMGB1 knockout HAP1 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 25 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab18256 observed at 29 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab18256 was shown to recognize HMGB1 in wild-type HAP1 cells as signal was lost at the expected MW in HMGB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. Ab18256 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Localisation and expression of HMGB1

    HMGB1 nuclear expression (brown staining) was seen in all pancreatic compartments, including stromal immune infiltrate: top panels show PanIN-1 (left) and -2 (right) (magnification ×50, insert and second panel x100); bottom panels show PanIN-3 (left) and PDAC (right) (magnification ×100 and ×50, respectively).

    PDCA = pancreatic adenocarcinoma. PanIN = precursor lesions.

    ab18256 is used at 1/1000 dilution.

    (After Figure 5 B of Crnongorac-Jurcevic et al)

  • ab18256 stained in Hela cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18256 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature

  • ab18256 used in Direct ELISA in NIH 3T3 murine fibroblasts. Primary antibody used at a 1/1000 dilution for 16 hours at 4°C. The secondary antibody is an AP-conjugated Goat anti-rabbit used at a 1/1000 dilution. A blocking step was performed using 5% BSA for 1 hour.

    See Abreview

  • ab18256 staining HMGB1 in Human stomach tissue sections by IHC-Fr (Immunohistochemistry - Frozen sections). Tissue samples were fixed with acetone and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody 1/500 in blocking buffer for 1 hour at 25°C. An undiluted HRP-conjugated Goat polyclonal to rabbit IgG was used as secondary antibody.

    See Abreview

  • All lanes : Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1 µg/ml

    Lane 1 : NIH 3T3 whole cell lysate (ab7179)
    Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 25 kDa
    Observed band size: 29 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 59 kDa. We are unsure as to the identity of these extra bands.

  • All lanes : Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat whole cell lysate (ab7899)
    Lane 3 : A431 whole cell lysate (ab7909)
    Lane 4 : HEK293 whole cell lysate (ab7902)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 25 kDa
    Observed band size: 29 kDa why is the actual band size different from the predicted?

  • Image courtesy of Human Protein Atlas

    Paraffin embedded sections of human liver were incubated with ab18256 (1/1000 dilution) for 30 minutes at room temperature. Heat induced antigen retrieval was performed in citrate buffer pH 6.

    ab18256 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further images can be found www.proteinatlas.org

  • ab18256 staining HMGB1 in mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 15% serum for 60 minutes at 20°C; antigen retrieval was by heat mediation. Samples were incubated with primary antibody (1/1000 in TBS) for 18 hours at 20°C. An Alexa Fluor® 647-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • All lanes : Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1/1000 dilution

    Lane 1 : Rat brain whole tissue lysate - infused with asf for 1 week
    Lane 2 : Rat brain whole tissue lysate - infused with LPS for 1 week
    Lane 3 : Rat brain whole tissue lysate - infused with acsf for 8 weeks
    Lane 4 : Rat brain whole tissue lysate - infused with LPS for 8 weeks
    Lane 5 : Rat brain whole tissue lysate - infused with LPS for 4 weeks
    Lane 6 : Rat brain whole tissue lysate -infused with LPS for 4 weeks, after 2 weeks of LPS infusion were treated with neramexane for the next 2 weeks
    Lane 7 : Rat brain whole tissue lysate - infused with LPS for 4 weeks, after 2 weeks of LPS infusion were treated with memantine for the next 2 weeks.

    Lysates/proteins at 40 µg per lane.

    Secondary
    All lanes : Biotinylated Goat anti-rabbit IgG

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 25 kDa


    Exposure time: 30 seconds

    See Abreview

  • Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1 µg/ml + Recombinant Human HMGB1 protein (ab56525) at 0.01 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 25 kDa


    Exposure time: 2 minutes
  • ab18256 staining HMGB1 in murine kidney tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    Tissue was fixed in formaldehyde and a heat mediated antigen retrieval step was performed using citrate EDTA buffer pH 6.2. Samples were then blocked, then incubated with ab18256 at a 1/1000 dilution for 1 hour. The secondary used was a goat anti-rabbit IgG conjugated to HRP at a 1/1000 dilution.

    See Abreview

References

This product has been referenced in:
  • Ma F  et al. Spinal macrophage migration inhibitory factor and high mobility group box 1 mediate persistent bladder pain. Neurosci Lett 699:54-58 (2019). Read more (PubMed: 30708129) »
  • Carr DF  et al. Serum and blister-fluid elevation and decreased epidermal content of high-mobility group box 1 protein in drug-induced Stevens-Johnson syndrome/toxic epidermal necrolysis. Br J Dermatol N/A:N/A (2019). Read more (PubMed: 30613954) »
See all 248 Publications for this product

Customer reviews and Q&As

41-50 of 123 Abreviews or Q&A

Answer

Thank you for your reply. Your protocol seems fine, there is no specific protocol for a Western Blot specifically from plasma and you should see good results using a standard WB protocol as you have done. Given your results with HMGB1 and actin I think what is happening is that an abundant plasma protein is obscuring other proteins, which apparently is common when using plasma samples. The solution to this is to use a column to filter this out. I have placed some information below, we don't actually sell these columns so you may have to do a Google search to find them. Please let me know if I can be of further assistance.

Depletion of high abundance protein from serum or plasma samples
When investigating plasma or serum by Western blotting, abundant plasma proteins, such
as albumin and IgG can obscure the signals of less abundant proteins. Prepacked columns,
such as HiTrap. Albumin & IgG Depletion are designed to deplete samples of these potentially
problematic proteins, removing >95% albumin and >90% IgG, respectively.
HiTrap Albumin & IgG Depletion 1 ml column is designed for the depletion of albumin and IgG
from sample volumes of approximately 150 ƒÊl of undiluted human plasma or serum, containing
normal levels of albumin (˜40 mg /ml) and IgG (˜15 mg/ml). The depletion procedure takes
approximately 35 min, and can be performed using a liquid chromatography system from the
AKTA. design platform, a peristaltic pump, or manually with a syringe. When working with
smaller volumes, Albumin & IgG Depletion SpinTrap., designed for volumes of ˜50 ƒÊl of human
plasma or serum, is recommended.

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Answer

Thank you for providing the details. Obviously these will help us in understanding the quality of this product.

We have never observed membrane like this, antibodies either shows multiple banding pattern or no bands in western blot. So before I replace the antibody I would like to ask if any step was changed in protocol as compared to best results of ab18256. Were the ECL reagents mixed properly? Is it possible if you

My suggestion for further improvement would be using highly diluted primary and secondary antibodies. Cutting membrane in half and probing with ab18256 and ab59463 would help in further investigation of the problem.

Finally would you like to receive replacement vial or product.

Many thanks for your cooperation I will look forward to hearing from you soon.

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Question

1) Abcam product code ab59463
2) Abcam order reference number or product batch number lot number 884532
3) Description of the problem: When developing the film with ECl reagens, the hole membrane is black on the film. 4) Sample preparation: Type of sample (whole cell lysates, fraction, recombinant protein…)
Epidermis whole cell lysate and cytosol and nucleus fractions from 3 different kind of squamous cell cancers
Lysis buffer LB buffer for the cytosolic proteins and 1xcell lysis for whole lysate and nucleus
Protease inhibitors: PMSF and COMPLETE Phosphatase inhibitors: PMSF and COMPLETE
Reducing agent: ? Boiling for ≥5 min? yes/no: no 3 min. Protein loaded ug/lane or cells/lane: 10 µg/lane
Positive control:no, but with HMGB1 ab18256 I get perfect western blots. Negative control: no
5) Percentage of gel: 3-12% Type of membrane: PVDF Protein transfer verified: yes Blocking agent and concentration: milk 5% in TBST Blocking time: 1 hour, Blocking temperature: room temperature 6) Primary antibody (If more than one was used, describe in “additional notes”) :Concentration or dilution: 1:1000 Diluent buffer : 5% BSA in TBST Incubation time: over night Incubation temperature: 4 degrees 7) Secondary antibody: Species: Goat Reacts against: rabbit Concentration or dilution: 1:2000 Diluent buffer : 5% milk in TBST Incubation time: 1 hour Incubation temperature: room temperature Fluorochrome or enzyme conjugate: HRP 8) Washing after primary and secondary antibodies: 3x 10 minutes in TBST+ 1x TBS before ECL is added Buffer: TBST Number of washes 9) Detection method: ECL 10) How many times have you run this staining? 4 Do you obtain the same results every time? yes What steps have you altered to try and optimize the use of this antibody? Test on whole lysate, and cytosol and nucleus. Have tried different dilutions of primary antibody and a secondary antibody from dako

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Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

I have read the details you have kindly provided and have following further questions for better understanding of the problem;

- Could you provide an image?
- Did you use same lysates with antibody ab18256?
- Are the ECL reagents OK?
- Was the PVDF membrane activated by soaking in methanol?
- What is the species of cell lysates used?

Thank you very much for your cooperation. I will look forward to hearing from you soon.

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Answer

Thank you for contactin us.

At Abcam we strive to share all non proprietary data publicly on the datasheets for our products. We try to not keep images which we may have available in archive as we feel that sharing all the information which we have about a product is the best way to help our customers find the product which suit their needs. While is extremely uncommon for us to withhold images I did review the datasheet for this product, including the Abreviews in hope that I may be able to put you in touch which a collaborator or customer how may have submitted these. However, I have not been able to locate the reference you have noted to staining in bone marrow. Is there perhaps another product to which you are referring?

I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for contacting us.

As shown on the product datasheets, ab9614 and ab18256 are bothpolyclonal antibodies. The same negative control should therefore besuitable for both products.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

As indicated on the product datasheet the observed molecular weight is ˜29 kDa.

To reduce the background, I would suggest:
- to load a maximum of 20µg of protein on the gel,
- to block the membrane for 1 hour with 5% BSA solution,
- to incubate the primary diluted 1/2000 or 1/1000in the blocking solution (5% BSA), overnight at 4ºC,
- to run a non-primary control to determine if some bands are due to the secondary.

I hope this information is helpful to you.

Please let me know if these suggestions help toimprove the results. If not, could you please ask your customer to fill in a questionnaire?

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Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Liver)
Specification
Liver
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH 6.0
Permeabilization
No
Blocking step
Protein block buffer read to use (Dako) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 26 2012

Application
Western blot
Sample
Mouse Tissue lysate - whole (Liver)
Loading amount
20 µg
Specification
Liver
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 26 2012

Application
Western blot
Sample
Human Tissue lysate - whole (Liver)
Loading amount
20 µg
Specification
Liver
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 26 2012

41-50 of 123 Abreviews or Q&A

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