Validated using a knockout cell line

Anti-Integrin beta 1 antibody [P5D2] (ab24693)

Reviews (3)Q&A (13)References (44)

Overview

  • Product name

    Anti-Integrin beta 1 antibody [P5D2]
    See all Integrin beta 1 primary antibodies

  • Description

    Mouse monoclonal [P5D2] to Integrin beta 1

  • Host species

    Mouse

  • Tested applications

    Suitable for: Blocking, ICC/IF, Flow Cyt, WB, IP, IHC-P, Electron Microscopymore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Tissue, cells or virus corresponding to Integrin beta 1. Tissue/ cell preparation (Human). Fibroblast cells (HT1080).

  • Positive control

    • ICC/IF: HEK293, HeLa cells. Flow cytometry: HEK293, MCF7 cells.

  • General notes

    Abcam recommends using this product under native conditions in western blot.  ab179471 is a possible alternative product for use in denaturing western blot.

    This monoclonal antibody to integrin beta 1 has been knockout validated in ICC/IF and flow cytometry. The expected signal was observed in wild type cells and no signal was seen in knockout cells.

    This antibody inhibits the function of beta 1 integrins and can be used to block cell adhesion (Dittell et al., 1993 and Yokosaki et al., 1994).

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab24693 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
Blocking Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 - 5 µg/ml.
Flow Cyt Use 0.1-1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB 1/1000. Predicted molecular weight: 88 kDa.

Abcam recommends using this product under native conditions in western blot.
IP 1/1000.
IHC-P Use at an assay dependent concentration. PubMed: 24705394
Electron Microscopy Use at an assay dependent concentration. PubMed: 24705394

Target

  • Function

    Integrins alpha-1/beta-1, alpha-2/beta-1, alpha-10/beta-1 and alpha-11/beta-1 are receptors for collagen. Integrins alpha-1/beta-1 and alpha-2/beta-2 recognize the proline-hydroxylated sequence G-F-P-G-E-R in collagen. Integrins alpha-2/beta-1, alpha-3/beta-1, alpha-4/beta-1, alpha-5/beta-1, alpha-8/beta-1, alpha-10/beta-1, alpha-11/beta-1 and alpha-V/beta-1 are receptors for fibronectin. Alpha-4/beta-1 recognizes one or more domains within the alternatively spliced CS-1 and CS-5 regions of fibronectin. Integrin alpha-5/beta-1 is a receptor for fibrinogen. Integrin alpha-1/beta-1, alpha-2/beta-1, alpha-6/beta-1 and alpha-7/beta-1 are receptors for lamimin. Integrin alpha-4/beta-1 is a receptor for VCAM1. It recognizes the sequence Q-I-D-S in VCAM1. Integrin alpha-9/beta-1 is a receptor for VCAM1, cytotactin and osteopontin. It recognizes the sequence A-E-I-D-G-I-E-L in cytotactin. Integrin alpha-3/beta-1 is a receptor for epiligrin, thrombospondin and CSPG4. Alpha-3/beta-1 may mediate with LGALS3 the stimulation by CSPG4 of endothelial cells migration. Integrin alpha-V/beta-1 is a receptor for vitronectin. Beta-1 integrins recognize the sequence R-G-D in a wide array of ligands. Isoform 2 interferes with isoform 1 resulting in a dominant negative effect on cell adhesion and migration (in vitro). When associated with alpha-7/beta-1 integrin, regulates cell adhesion and laminin matrix deposition. Involved in promoting endothelial cell motility and angiogenesis. Involved in osteoblast compaction through the fibronectin fibrillogenesis cell-mediated matrix assembly process and the formation of mineralized bone nodules. May be involved in up-regulation of the activity of kinases such as PKC via binding to KRT1. Together with KRT1 and RACK1, serves as a platform for SRC activation or inactivation. Plays a mechanistic adhesive role during telophase, required for the successful completion of cytokinesis. Integrin alpha-3/beta-1 provides a docking site for FAP (seprase) at invadopodia plasma membranes in a collagen-dependent manner and hence may participate in the adhesion, formation of invadopodia and matrix degradation processes, promoting cell invasion. ITGA4:ITGB1 binds to fractalkine (CX3CL1) and may act as its coreceptor in CX3CR1-dependent fractalkine signaling (PubMed:23125415, PubMed:24789099). ITGA4:ITGB1 and ITGA5:ITGB1 bind to PLA2G2A via a site (site 2) which is distinct from the classical ligand-binding site (site 1) and this induces integrin conformational changes and enhanced ligand binding to site 1 (PubMed:18635536, PubMed:25398877). ITGA5:ITGB1 acts as a receptor for fibrillin-1 (FBN1) and mediates R-G-D-dependent cell adhesion to FBN1 (PubMed:12807887, PubMed:17158881).
    Isoform 5: Isoform 5 displaces isoform 1 in striated muscles.
    (Microbial infection) Integrin ITGA2:ITGB1 acts as a receptor for human echoviruses 1 and 8 (PubMed:8411387). Acts as a receptor for cytomegalovirus/HHV-5 (PubMed:20660204). Acts as a receptor for Epstein-Barr virus/HHV-4 (PubMed:17945327). Integrin ITGA5:ITGB1 acts as a receptor for human parvovirus B19 (PubMed:12907437). Integrin ITGA2:ITGB1 acts as a receptor for human rotavirus (PubMed:12941907). Acts as a receptor for mammalian reovirus (PubMed:16501085). In case of HIV-1 infection, integrin ITGA5:ITGB1 binding to extracellular viral Tat protein seems to enhance angiogenesis in Kaposi's sarcoma lesions (PubMed:10397733).

  • Tissue specificity

    Isoform 1 is widely expressed, other isoforms are generally coexpressed with a more restricted distribution. Isoform 2 is expressed in skin, liver, skeletal muscle, cardiac muscle, placenta, umbilical vein endothelial cells, neuroblastoma cells, lymphoma cells, hepatoma cells and astrocytoma cells. Isoform 3 and isoform 4 are expressed in muscle, kidney, liver, placenta, cervical epithelium, umbilical vein endothelial cells, fibroblast cells, embryonal kidney cells, platelets and several blood cell lines. Isoform 4, rather than isoform 3, is selectively expressed in peripheral T-cells. Isoform 3 is expressed in non-proliferating and differentiated prostate gland epithelial cells and in platelets, on the surface of erythroleukemia cells and in various hematopoietic cell lines. Isoform 5 is expressed specifically in striated muscle (skeletal and cardiac muscle).

  • Sequence similarities

    Belongs to the integrin beta chain family.
    Contains 1 VWFA domain.

  • Post-translational
    modifications

    The cysteine residues are involved in intrachain disulfide bonds.

  • Cellular localization

    Cell membrane, sarcolemma. Cell junction. In cardiac muscle, isoform 5 is found in costameres and intercalated disks and Cell membrane. Cell projection, invadopodium membrane. Cell projection, ruffle membrane. Recycling endosome. Melanosome. Cleavage furrow. Cell projection, lamellipodium. Cell projection, ruffle. Cell junction, focal adhesion. Cell surface. Isoform 2 does not localize to focal adhesions. Highly enriched in stage I melanosomes. Located on plasma membrane of neuroblastoma NMB7 cells. In a lung cancer cell line, in prometaphase and metaphase, localizes diffusely at the membrane and in a few intracellular vesicles. In early telophase, detected mainly on the matrix-facing side of the cells. By mid-telophase, concentrated to the ingressing cleavage furrow, mainly to the basal side of the furrow. In late telophase, concentrated to the extending protrusions formed at the opposite ends of the spreading daughter cells, in vesicles at the base of the lamellipodia formed by the separating daughter cells. Colocalizes with ITGB1BP1 and metastatic suppressor protein NME2 at the edge or peripheral ruffles and lamellipodia during the early stages of cell spreading on fibronectin or collagen. Translocates from peripheral focal adhesions sites to fibrillar adhesions in a ITGB1BP1-dependent manner. Enriched preferentially at invadopodia, cell membrane protrusions that correspond to sites of cell invasion, in a collagen-dependent manner. Localized at plasma and ruffle membranes in a collagen-independent manner.
  • Information by UniProt

  • Database links

    • Alternative names

      • beta1 integrin antibody
      • CD_antigen=CD29 antibody
      • CD29 antibody
      • Fibronectin receptor subunit beta antibody
      • FNRB antibody
      • Glycoprotein IIa antibody
      • GP IIa antibody
      • GPIIA antibody
      • Integrin beta-1 antibody
      • Integrin subunit beta 1 antibody
      • integrin VLA-4 beta subunit antibody
      • Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) antibody
      • ITB1_HUMAN antibody
      • ITGB1 antibody
      • MDF2 antibody
      • MSK12 antibody
      • OTTHUMP00000019420 antibody
      • very late activation protein antibody
      • Very late activation protein, beta polypeptide antibody
      • VLA BETA antibody
      • VLA-4 subunit beta antibody
      • VLA-BETA antibody
      • VLAB antibody
      • VLAbeta antibody
      see all

    Images

    • Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)

      ab24693 staining Integrin beta 1 in wild-type HAP1 cells (top panel) and Integrin beta 1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab24693 at 5μg/ml concentration and ab202272 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    • Flow Cytometry - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Flow Cytometry - Anti-Integrin beta 1 antibody [P5D2] (ab24693)

      Overlay histogram showing HAP1 wildtype (green line) and HAP1-ITGB1 knockout cells (red line) stained with ab24693. Live HAP1 wildtype and HAP1-ITGB1 knockout cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab24693, 0.1µg/0.5x106 cells) for 30 min at 22°C. A mouse IgG1 isotype control antibody  (ab170190) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-ITGB1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

    • Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)Image from Modulevsky DJ et al., PLoS One. 2012;7(9):e45329. Fig 7.; doi: 10.1371/journal.pone.0045329. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

      Cells were exposed to inhibitors for 60 min and then fixed and stained for the presence of β1-integrin (greyscale and red in the merge) and the plasma membrane (green in the merge). LSCM images were acquired at the cell-substrate interface. (A) Control (scale bar = 20 µm and applies to all), (B) Cyt-D, (C) nocodazole, (D) Y-27632A, (E) ML-7 and (F) blebbistatin treated cells. In all cases, integrin-ß1 is well distributed over the cell contact area. However, after 60 min of CytD treatment a significant decrease in integrin-ß1 was observed, correlating to a significant decrease in cell adhesion strength (Fig. 5).

      Cells were first fixed by parafromaldehyde and subsequently extracted by methanol. ab24693 and Alexa Fluor® 546 rabbit anti-mouse immunoglobin were used as primary and secondary antibodies, respectively. Cells were also stained with wheat germ agglutinin coupled to Oregon Green 488 to reveal the cell membrane.

    • Flow Cytometry - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Flow Cytometry - Anti-Integrin beta 1 antibody [P5D2] (ab24693)

      Overlay histogram showing MCF7 cells stained with ab24693 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab24693, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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 antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    • Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)This image is courtesy of an anonymous abreview.

      ICC/IF image of ab24693 stained HeLa cells. The cells were formaldehyde fixed, permeabilization with 0.5% TritonX/PBS and then blockedwith 5% BSA for 30 minutes at 21°C. The cells were then incubated with the antibody (ab24693, 1/200) for 1 hour at 21°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/500 dilution. DAPI was used to stain the cell nuclei (blue) and Phalloidin-TRITC (red) the actin cytoskeleton.

      See Abreview

    • Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Immunocytochemistry/ Immunofluorescence - Anti-Integrin beta 1 antibody [P5D2] (ab24693)Image from Molladavoodi S et al., PLoS One. 2017;12(6):e0178981. Fig 8.; doi: 10.1371/journal.pone.0178981. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

      Higher intensity for integrin β1 staining can be observed in the low shear sample compared to no shear.

      a) Control (no shear) sample, b) After 24 hours of low shear stress exposure.

      Control cells and cells exposed to low shear for 24-hr were fixed with 4% paraformaldehyde. Samples were permeabilized with 0.1% Triton and nonspecific binding was blocked with 1% BSA/10% goat serum. Samples were incubated with ab24693 followed by Alexa Fluor® 488 conjugated secondary antibody (ab150113).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin beta 1 antibody [P5D2] (ab24693)Image from Yuan X et al., PLoS One. 2014;9(4):e94212. Fig 2.; doi: 10.1371/journal.pone.0094212.

      Immunohistochemical localization of podocyte proteins in kidney tissues.

      The tissues were prepared with IVCT and immersion-fixation methods under various hemodynamic conditions. The micrographs of A, B, C, and D show the localization of integrin β1; F, G, H and I exhibit control stainings ommitting the integrin β1 primary antibody.

      Immunohistochemical localizations are shown under normotensive condition (A), acute hypertensive condition (B), and cardiac arrest condition (C). The tissue in D was treated by the immersion-fixation method.

      Scale bars  = 20 μm.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin beta 1 antibody [P5D2] (ab24693)Image from Yuan X et al., PLoS One. 2014;9(4):e94212. Fig 3.; doi: 10.1371/journal.pone.0094212.

      Confocal laser scanning micrographs show the double-fluorescence of integrin β1-WT1.

      The double-staining micrographs are shown in L, O, and R (integrin β1 and WT1, arrows). Both of them were distributed in erythrocytes (asterisk). Left bottom pictures are the manified micrographs.

      Scale bars  = 20 μm.

       

    • Electron Microscopy - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Electron Microscopy - Anti-Integrin beta 1 antibody [P5D2] (ab24693)Image from Yuan X et al., PLoS One. 2014;9(4):e94212. Fig 4.; doi: 10.1371/journal.pone.0094212. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

      Immune electron micrographs of integrin β1 in glomeruli under normotensive and acute hypertensive conditions.

      Under normotensive condition, the foot processes of podocytes were shown to be shrunken with the conventional fixation method (F). In contrast, using IVCT, the foot processes tightly approached each other and became flatter, integrin β1 was distributed on the basolateral membrane of podocytes (D). In contrast, under hypertensive condition, integrin β1 attached along the basal membrane only (E). Left bottom pictures are the manified micrographs. They were magnified 10 K. (Magnification, ×6000 for D; ×4000 for E; ×10 K for F).

       
    • Western blot - Anti-Integrin beta 1 antibody [P5D2] (ab24693)
      Western blot - Anti-Integrin beta 1 antibody [P5D2] (ab24693)This image is courtesy of an anonymous Abreview.
      All lanes : Anti-Integrin beta 1 antibody [P5D2] (ab24693) at 1/1000 dilution

      Lane 1 : Human melanoma whole cell lysate - shGFP
      Lane 2 : Human melanoma whole cell lysate - shMT1-MMP
      Lane 3 : Human melanoma whole cell lysate - shGFP+Integrin beta1 overexpression
      Lane 4 : Human melanoma whole cell lysate - shMT1-MMP+Integrin beta1 overexpression

      Lysates/proteins at 30 µg per lane.

      Secondary
      All lanes : Anti-mouse IgG, HRP-linked antibody at 1/1 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 88 kDa
      Observed band size: 130 kDa
      why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      10% gel. Blocked with 5% milk for 1 hour at 25°C. Incubated with the primary antibody for 18 hours at 4°C.

      See Abreview

    References

    This product has been referenced in:

    See all 44 Publications for this product

    Publishing research using ab24693? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-10 of 16 Abreviews or Q&A

    Western blot abreview for Anti-Integrin beta 1 antibody [P5D2]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (melanoma)
    Gel Running Conditions
    Reduced Denaturing (10%)
    Loading amount
    30 µg
    Specification
    melanoma
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 28 2019

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Integrin beta 1 antibody [P5D2]

     Good
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 21°C
    Sample
    Human Cell (HeLa cell)
    Specification
    HeLa cell
    Permeabilization
    Yes - 0.5% TritonX/PBS
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 19 2013

    Question

    Link to antibody purification kit.

    Read More
    Answer

    Thank you for contacting Abcam.

    Please find the link to the antibody purification kit below, that will allow you to remove the sodium azide from your antibody solution:

    https://www.abcam.com/antibody-purification-kit-protein-a-ab102784.html



    If there is anything else I can help you with, just let me know.

    Read More

    Question

    I would like to follow up on the replacement antibody discussed below.

    Can we request a replacement?

    Regards,

    Read More
    Answer

    Thank you very much for your email.

    Looking through our records, it seems we already issued a credit to compensate for the ab30394 that wasn't working, as requested by email in May. The credit note number is ***, and it was probably applied back to the credit card used on the order. Could you confirm with your purchasing department whether this credit was received? They can contact our accounting department at mailto:us.credits@abcam.com or 888-772-2226 if they have any questions. This credit can be applied to the invoice of a new order placed with us, so it will essentially be a replacement antibody of your choice.

    I am very sorry for any confusion regarding this credit note, but please let me know if you have any questions or concerns, and I'll be happy to help you further.

    Read More

    Question

    I know that this antibody is specific for integrin beta 1. I want to know if it will bind to only to activated integrin beta 1 or it will bind to all integrin beta 1 (activated or not).

    Read More
    Answer

    Thank you for your reply.


    My apologies! The antibody will recognize all integrin beta 1 and is not specific for activated integrin beta 1.


    Please let me know if you have any additional questions!

    Read More

    Question

    We have Anti-integrin beta 1 antibody [P5D2]. Is it specific for the activated state of beta 1 integrin or it binds to all integrin proteins

    Read More
    Answer

    Thank you for contacting Abcam regarding ab24693.


    This antibody is specific for integrin beta 1 and is not expected to react with all integrin proteins.


    I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

    Read More

    Question

    Hi Kate,

    According to our records, our details are as follows;

    Our PO### was dated ###

    Looking forward to your response.

    Kind regards,

    Read More
    Answer

    Thank you for kindly confirming these details. I am sorry this vial of antibody has not worked for this customer.

    As requested, we are pleased to arrange a free of charge replacement. I can confirm that 1 vial of alternative replacement ab52971 has been added to your order number ######. This has Abcam order reference #####.

    I would like to reassure you that this free of charge replacement vial is also covered by our Abpromise guarantee. Should the customer still be experiencing difficulties with the new vial, or if you have any further questions, please do not hesitate to let me know.

    Also, please note that this item was purchased 8 months ago, outside our guarantee period of 6 months. Therefore this free of charge replacement is being provided as an exception in this case. We would encourage all customers to please contact us as soon as possible if they encounter any difficulties, and within 6 months of purchase to be covered by the guarantee.

    Thank you for your help and cooperation with this case. Please do not hesitate to contact me if you need anything further.

    Read More

    Question

    Dear Abcam Technical Support Team,

    A customer contacted us in response to: AB24693 (Anti-Integrin beta 1 antibody [P5D2])

    This product was originally purchased by the customer for use on mouse tissue via Western blot.

    Western blots experiments were carried out on mouse tissue between January-May 2012, but there were issues with the results (see the attached Technical Enquiry Form).

    When this product was purchased in Oct 2011, the product datasheet sheet mentioned the species reactivity was: human, mouse (see the attached datasheet from Jan 2012)

    In May, the datasheet appears to have changed for species reactivity: human (see the attached datasheet from May 2012)

    The customer would like to know why the ‘mouse’ species reactivity was recently removed from this product datasheet and whether this could be the reason her experiments are not working?

    Kind regards,


    ANTIBODY DETAILS
    Antibody Code:AB24693
    Batch Number:GR654-6
    GENERAL INFORMATION
    Antibody Storage Conditions (temperature/reconstitution etc)
    Aliquotted and stored at -20oC
    Description of the Problem (high background, wrong band size, more bands, no bands etc)
    Very faint signal
    Bands of incorrect size (50-60kda)
    When the ab. was purchased the data sheet stated that the ab. has species reactivity with mouse, but now the data sheet has been updated and only stated cross reactivity with human
    Sample (Species/Cell extract/Purified protein/Recombinant protein, etc)
    Mouse tissue total protein extract
    Sample Preparation (Buffer/Protease inhibitors/Heating sample etc.)
    Extracted using RIPA Lysis and Extraction Buffer (Thermo #89901) with added protease inhibitors
    Amount of protein loaded
    10ug – 20ug
    Electrophoresis/Gel Conditions (Reducing or non-reducing gel, % of the gel etc.)
    10% acrylamide or 4-15% gradient acrylamide gels
    Reducing conditions
    Transfer and blocking conditions (Buffer/time period, Blocking agent etc)
    Transfer Buffer 25mM Tris, 192mM Glycine, 20% MeOH, pH 8.3
    Transferred for 60 min at 90V, transfer assessed by Ponceau Red staining
    Blocked O/N at 4oC in either 5% Skim milk/TBST or 2% BSA/PBST
    Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
    Abcam ab24693 diluted 1:1000 in 2% Skim milk/TBST or 1% BSA/PBST for 2h @ RT
    Washed 3 x 10 min in TBST
    Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
    Rabbit Anti-Mouse IGG/HRP (Sigma A9044) diluted 1:1000 in 2% Skim milk/TBST or 1% BSA/PBST for 2h @ RT
    Washed 3 x 10 min in TBST
    Detection Method (ECL, ECL Plus etc.)
    SuperSignal West Pico Chemiluminescent Substrate (Thermo #34080) for 5min at RT
    Positive and negative controls used (please specify)
    Samples from diseased animal models showing significant tissue inflammation were compared to tissues from control animal models (negative control)
    Blots are probed with beta actin as a loading control (positive control)
    OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)
    How many times have you tried the Western Blot?
    5
    Have you run a “No Primary” control? No
    Do you obtain the same results every time? Yes
    e.g. are the background bands always in the same place?
    What steps have you altered?
    Increased ab. concentration
    Increased length of ab. probing to O/N at 4oC
    Increased protein concentration
    Varied the blocking and diluent from skim milk/TBST to BSA/PBST
    Varied the range of tissue samples used
    Purchased a new secondary ab.
    Purchased new detection reagents
    Confirmed blot results on two different imaging systems: ChemiDoc EQ (Bio-Rad) or ImageQuant LAS 4000 Mini (GE Healthcare)
    Additional Notes

    Read More
    Answer

    Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

    The details you have kindly provided will provide us with vital information for our monitoring of product quality

    This antibody used to be sourced externally, and had been successfully tested in mouse. After the customer purchased this product, from November last year,we now producethe same clone in house from the same hybridoma,but it has not yet been tested by us in mouse samples so mouse was removed. So when the customer purchased the antibody, it was guaranteed in mouse.

    My colleagues are currently checking the data from a publication in which the same clone has been used in mouse to see whether we can add this speciesagain.

    I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

    I apologize for the inconvenience.Although thisitem was purchased over6 months ago,I am pleased to make an exceptionin this case and offer a free of charge replacement or credit note in compensation. In order to arrange this, I would appreciate if you could please confirm the order number

    However, please note that we would encourage customers to contact us as soon as possible if they encounter any difficulties and to contact us within 6 months.

    In addition in this case, I can suggest you may like to consider the following suggestions as a check for future experiments:

    1. I suggest to try blocking for 1 hour at room temperature. Overnight may be too long.

    2. Was the sample heated to 95oC for 5 minutes in sample buffer containing SDS and mercaptoethanol to reduce and denature? This will help to ensure the protein is in the correct conformation to run at the correct molecular weight.

    Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

    Read More

    Question

    can you issue an abcam credit to the account for the amount so we can order what we need off it when we need stuff? We need 1 antibody now that’s a secondary – but if you issue a full credit, I am fearful the funds won’t go back towards getting antibodies ;).



    If this is possible, let me know. Or If you can order the replacement for us, and then put the additional as a credit on our abcam account – that would be great.

    Read More
    Answer

    Thank you for your reply, and I'm sorry we couldn't get this antibody working for you.

    I've gone ahead and issued the full credit for your original purchase, and since this was paid by credit card it will actually be refunded back to your card pending approval of our accounting department. You can contact our accounting department at mailto:us.credits@abcam.com if you have any questions about the credit, and please referenced credit note # ***.

    I am sorry but our system isn't set up so that I can send a replacement for a cheaper item and then credit the difference, otherwise I would have done that (I know that would have been easier for you!). But you can go ahead and use your card to order the secondary antibody and the finances should even out. All of our secondary antibodies are 25% off during the month of May, so that may be incentive to go ahead and order the one you need :)

    Please let me know if you need anything else from me, and I'll be happy to help! Have a great day.

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    Question

    thank you for being patient with us in trying to get an antibody working. I was wondering what the process was like to get a replacement product for the cost of the B1 antibody, but not the same product?

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    Answer

    Thank you for your email. I apologize again for all of the trouble with this antibody. I'd be happy to send any antibody you'd like as a replacement. If there is a cost discrepancy, I can issue a credit for your original puchase, and you can order the replacement item separately, which will even out the prices.

    Which product would you like as a replacement, and I will see which option would be best?

    Please let me know if you have any further questions. I look forward to hearing from you.

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