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  1. Link

    pyk2-antibody-ye353-ab32571.pdf

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Signal Transduction Protein Phosphorylation Tyrosine Kinases FAK / PYK
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-PYK2 antibody [YE353] (ab32571)

  • Datasheet
Submit a review Q&A (4)References (25)

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Western blot - Anti-PYK2 antibody [YE353] (ab32571)
  • Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (ab32571)
  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
  • Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (ab32571)
  • Anti-PYK2 antibody [YE353] (ab32571)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [YE353] to PYK2
  • Suitable for: WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647 Carrier Free HRP

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Overview

  • Product name

    Anti-PYK2 antibody [YE353]
    See all PYK2 primary antibodies
  • Description

    Rabbit monoclonal [YE353] to PYK2
  • Host species

    Rabbit
  • Specificity

    This antibody recognizes PYK2. It does not cross react with other FAK family members.
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human PYK2 aa 1-100 (N terminal). The exact sequence is proprietary.
    Database link: Q14289

  • Positive control

    • WB: Ramos, Jurkat and RAW264.7 cell lysates and mouse and rat brain tissue lysates. IHC-P: Human, mouse and rat cerebral cortex tissues. ICC/IF: HeLa and PC12 cells.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    YE353
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • FAK / PYK
    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Other
    • Cancer
    • Cancer Metabolism
    • Response to hypoxia
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia
    • Metabolism
    • Types of disease
    • Cancer

Associated products

  • Alternative Versions

    • HRP Anti-PYK2 antibody [YE353] (ab214497)
    • Alexa Fluor® 647 Anti-PYK2 antibody [YE353] (ab224955)
    • Alexa Fluor® 488 Anti-PYK2 antibody [YE353] (ab224956)
    • Anti-PYK2 antibody [YE353] - BSA and Azide free (ab228477)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • Ramos whole cell lysate (ab3955)
    • RAW 264.7 whole cell lysate (ab7187)
    • Mouse brain tissue lysate - total protein (0 days) (ab7188)
    • Mouse brain tissue lysate - total protein (14 days) (ab7189)
    • Mouse brain tissue lysate - total protein (7 days) (ab7190)
    • Jurkat whole cell lysate (ab7899)
  • Recombinant Protein

    • Recombinant Human PYK2 protein (ab152380)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32571 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
1/2000. Detects a band of approximately 116 kDa (predicted molecular weight: 116 kDa).

For unpurified use at 1/1000 - 1/5000.

IHC-P
1/300. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/250 - 1/500.

ICC/IF
1/60.

For unpurified use at 1/100.

Notes
WB
1/2000. Detects a band of approximately 116 kDa (predicted molecular weight: 116 kDa).

For unpurified use at 1/1000 - 1/5000.

IHC-P
1/300. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/250 - 1/500.

ICC/IF
1/60.

For unpurified use at 1/100.

Application notes
Is unsuitable for IP.

Target

  • Function

    Involved in calcium induced regulation of ion channel and activation of the map kinase signaling pathway. May represent an important signaling intermediate between neuropeptide activated receptors or neurotransmitters that increase calcium flux and the downstream signals that regulate neuronal activity. Interacts with the SH2 domain of Grb2. May phosphorylate the voltage-gated potassium channel protein Kv1.2. Its activation is highly correlated with the stimulation of c-Jun N-terminal kinase activity. Involved in osmotic stress-dependent SNCA 'Tyr-125' phosphorylation. In concert with SRC, plays an important role in osteoclastic bone resorption. Both the formation of a SRC-PTK2B complex, and SRC kinase activity are necessary for this function. The Tyr-402 phosphorylated form serves as a docking site for SRC and is important for the organization of the osteoclast actin cytoskeleton and attachment sites and for bone resorption.
  • Tissue specificity

    Most abundant in the brain, with highest levels in amygdala and hippocampus. Low levels in kidney. Also expressed in spleen and lymphocytes.
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. FAK subfamily.
    Contains 1 FERM domain.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications

    Phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by PKC activation. Recruitment by nephrocystin to cell matrix adhesions initiates Tyr-402 phosphorylation. In monocytes, adherence to substrata is required for tyrosine phosphorylation and kinase activation. Angiotensin II, thapsigargin and L-alpha-lysophosphatidic acid (LPA) also induce autophosphorylation and increase kinase activity.
  • Cellular localization

    Cytoplasm. Cell membrane. Interaction with nephrocystin induces the membrane-association of the kinase.
  • Target information above from: UniProt accession Q14289 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 2185 Human
    • Entrez Gene: 19229 Mouse
    • Entrez Gene: 50646 Rat
    • Omim: 601212 Human
    • SwissProt: Q14289 Human
    • SwissProt: Q9QVP9 Mouse
    • SwissProt: P70600 Rat
    • Unigene: 491322 Human
    • Unigene: 21613 Mouse
    • Unigene: 11025 Rat
    see all
  • Alternative names

    • CADTK antibody
    • CAK-beta antibody
    • CAKB antibody
    • CAKbeta antibody
    • Calcium regulated non receptor proline rich tyrosine kinase antibody
    • Calcium-dependent tyrosine kinase antibody
    • Cell adhesion kinase beta antibody
    • E430023O05Rik antibody
    • EC 2.7.10.2 antibody
    • FADK 2 antibody
    • FADK2 antibody
    • FAK2 antibody
    • FAK2_HUMAN antibody
    • Focal adhesion kinase 2 antibody
    • MGC124628 antibody
    • PKB antibody
    • Proline-rich tyrosine kinase 2 antibody
    • Protein kinase B antibody
    • Protein Tyrosine Kinase 2 Beta antibody
    • Protein-tyrosine kinase 2-beta antibody
    • PTK antibody
    • PTK2B antibody
    • PTK2B protein tyrosine kinase 2 beta antibody
    • PYK2 antibody
    • RAFTK antibody
    • RAFTK2 antibody
    • Related adhesion focal tyrosine kinase antibody
    see all

Images

  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Western blot - Anti-PYK2 antibody [YE353] (ab32571)

    Lane 1: Wild-type HAP1 whole cell lysate (20 µg)              
    Lane 2: PYK2 knockout  HAP1 whole cell lysate (20 µg)
    Lane 3: Jurkat whole cell lysate (20 µg)
    Lane 4: Hu brain whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab32571 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab32571 was shown to specifically recognize PYK2 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when PYK2 knockout samples were examined. Wild-type and PYK2 knockout samples were subjected to SDS-PAGE.  Ab32571 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (ab32571)
    Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (ab32571)

    Immunocytochemistry/Immunofluorescence analysis of PC12 cells labelling PYK2 with unpurified ab32571 at a 1/100 dilution.

  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    All lanes : Anti-PYK2 antibody [YE353] (ab32571) at 1/10000 dilution (purified)

    Lane 1 : Ramos cell lysate
    Lane 2 : Jurkat cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 116 kDa
    Observed band size: 116 kDa



    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Anti-PYK2 antibody [YE353] (ab32571) at 10000 cells (purified) + RAW264.7 cell lysate at 20 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 116 kDa
    Observed band size: 116 kDa



    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    All lanes : Anti-PYK2 antibody [YE353] (ab32571) at 1/2000 dilution (purified)

    Lane 1 : Mouse brain tissue lysate
    Lane 2 : Rat brain tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 116 kDa
    Observed band size: 116 kDa



    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Western blot - Anti-PYK2 antibody [YE353] (ab32571)
    Anti-PYK2 antibody [YE353] (ab32571) at 1/5000 dilution (unpurified) + Jurkat cell lysate

    Predicted band size: 116 kDa
    Observed band size: 116 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling PYK2 with purified ab32571 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebral cortex tissue labelling PYK2 with purified ab32571 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue labelling PYK2 with purified ab32571 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (ab32571)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human brain tissue labelling PYK2 with unpurified ab32571 at a 1/250 dilution.

  • Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (ab32571)
    Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (ab32571)

    Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling PYK2 with purified ab32571 at 1/60. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

    Control: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

  • Anti-PYK2 antibody [YE353] (ab32571)
    Anti-PYK2 antibody [YE353] (ab32571)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • Datasheet download

    Download

References (25)

Publishing research using ab32571? Please let us know so that we can cite the reference in this datasheet.

ab32571 has been referenced in 25 publications.

  • Lee S  et al. Pyk2 Signaling through Graf1 and RhoA GTPase Is Required for Amyloid-ß Oligomer-Triggered Synapse Loss. J Neurosci 39:1910-1929 (2019). PubMed: 30626696
  • Jiang R  et al. The emerging roles of a novel CCCH-type zinc finger protein, ZC3H4, in silica-induced epithelial to mesenchymal transition. Toxicol Lett 307:26-40 (2019). PubMed: 30826420
  • Zhang Y  et al. Silencing Nogo-B receptor inhibits penile corpus cavernosum vascular smooth muscle cell apoptosis of rats with diabetic erectile dysfunction by down-regulating ICAM-1. PLoS One 14:e0220715 (2019). PubMed: 31442237
  • Tiede S  et al. The FAK inhibitor BI 853520 exerts anti-tumor effects in breast cancer. Oncogenesis 7:73 (2018). PubMed: 30237500
  • Roy NH  et al. Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1. Sci Signal 11:N/A (2018). PubMed: 30538176
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-4 of 4 Abreviews or Q&A

Question

I hope you are well.

Here are customer's answers:

- Could you please specify the cell types and the species of the samples? MDA-MB-134 and T47D cell lines are breast ductal carcinoma cell lines originated from Human
- Were the samples paraffin-embedded sections from tissues? No, the samples are paraffin-embedded cultured cells
- If not and the samples are from cultured cells why HIER was applied? I do not know what “HIER” means, anyway, this calibration was for the use in tissue samples later on, so we wanted to make the same condition as much as we can
- 20% Serum (normal horse serum) is very high particularly for blocking cells, normally 10% serum or 5% BSA is recommended. If the blocking is too stringent, it can significantly reduce the specific signal. 20% serum is the condition we are using routinely, we never experienced ‘the concentration of the serum’ making any problems.

- Generally, PFA fixation does not require antigen retrieval. In fact demasking can destroy the epitope after PFA- fixation. Please confirm if the fixation is 4% PFA or formalin fixed (10% NBF – neutral buffered formalin which is 4% formaldehyde)? 4% PFA, Please note that the figure of IHC we have provided, we know that the antigen is not destroyed in this condition. IHC with our antibodies show significant increase in the positive control (T47D). We were looking for the Pyk2 antibody which has strong signal without background. ab32571 satisfied us in term of ‘without background’, however, the signal intensity is much less than we have expected.

Thanks in advance fro your reply.

Have a nice day,

Read More

Abcam community

Verified customer

Asked on Dec 20 2012

Answer

Thank you for your kind reminder and your patience. I have been discussing this enquiry with some colleagues.
From what we understand the customer is using this antibody (ab32571: Anti-PYK2 antibody) for cell staining, so I would recommend our ICC protocol - please see the details at the bottom of this message.
The major changes would be to switch to 2% PFA fixation, and omit the antigen retrieval step. They could also modify the blocking step to only 10% serum in PBS for 1 hour (20% serum for cells is very high).
1. Solutions and reagents
1.1. Washing buffer:
PBST washing buffer: 1X PBS / 0.1% Tween-20 (Dulbeccos Phosphate Buffered Saline)
1.2. 2% Paraformaldehyde (prepare fresh by dissolving paraformaldehyde in 1X PBS by heating at 70°C until dissolving. Use cold.)
1.3. 0.1% Triton X-100
1.4. Blocking buffer:
PBS (Dulbeccos Phosphate Buffered Saline) + 10% serum (serum origin depends on the host of the secondary antibody)
1.5. Mounting medium for fluorescence.
1.6. Acetone (Production Grade).
2. Protocol
2.1. Grow cells in chamber slides or in 6-well tissue plates containing sterile coverslips. Prior to fixation, cells should not reach more than 80% confluency.
2.2. Fixation
2.2.1. Remove medium from chamber slides or 6-well plates. Wash once with PBS-T.
2.2.2. Fix cells with 2% paraformaldehyde for 20 min. For HeLa cells only, fix and permeabilize with acetone at -20°C for 5 minutes and continue to step 2.2.5.
2.2.3. Wash cells twice with 1XPBST.
2.2.4. Permeabilize cells with 0.1% Triton-X100 for 5 min
2.2.5. Wash cells twice with 1XPBST.
2.3. Blocking
2.3.1. Block cells with blocking buffer for 1 hour at room temperature (option - O/N 4°C).
2.4. Staining
2.4.1. Dilute primary antibody in the blocking buffer per recommendation on the data sheet.
2.4.2. Apply primary antibody on the cells and incubate overnight at 4°C.
2.4.3. Wash cells twice with 1XPBST.
2.4.4. Incubate cells in a dilution of fluorescently-labeled secondary antibody in PBS for 45 min at room temperature in the dark.
2.4.5. Wash cells three times with 1XPBST.
2.4.6. Counterstain cells with DAPI in a concentration of 300 nM in PBS for 5 min in the dark.
2.4.7. Wash cells three times with 1XPBST.
2.4.8. Mount slides with medium for fluorescent staining.
2.4.9. Store slides in the dark.
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.

Read More

Abcam Scientific Support

Answered on Dec 20 2012

Question

Dear Tech Support Team,

Please read below the details of customer's complaint and advise.

Attached are the images.

Antibody code: ab32571

Batch number: Lot GR386-9

General Information:

Antibody storage conditions -20˚C, in aliquot

Description of the problem low signal

Sample Paraffin section prepared from cell line

Fixation of sample Paraformaldehyde 4%

Antigen retrieval Microwave in Sodium Citrate solution (pH 6.0)

Permeabilization step 0.2% Triton-X-100 in blocking

Blocking conditions Normal Horse Serum 20 %, 1.5 h

Primary Antibody Normal Horse Serum 2 % in PBS, 0.5% Triton-X-100, Room temperature, Over-night, Washing : 3 times with PBS for each 3 min

Secondary Antibody Vectastain ABC kit RaBBIT IgG PK-6101/donkey anti-rabbit/Normal Horse Serum 2% in PBS, /1:200/Room Temperature 90 min, 3 times with PBS for each 3 min

Detection method DAB staining

Positive and negative controls used MDA-MB-134 cells for negative control, T47D for positive control

Optimization attempts (problem solving): higher concentration, up to 1:25

How many times have you tried the IHC? Twice

Have you run a "No Primary" control? No

Do you obtain the same results every time? Yes

What steps have you altered? Primary antibody dilution


Thanks in advance for your reply.

Have a nice week.

Read More

Abcam community

Verified customer

Asked on Dec 03 2012

Answer

Thank you for your enquiry regarding ab32571 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.
We have been selling this antibody without any problem so we are not aware of any quality-related issues.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
- Could you please specify the cell types and the species of the samples?
- Were the samples paraffin-embedded sections from tissues?
- If not and the samples are from cultured cells why HIER was applied?
- 20% Serum (normal horse serum) is very high particularly for blocking cells, normally 10% serum or 5% BSA is recommended. If the blocking is too stringent, it can significantly reduce the specific signal.
Generally, PFA fixation does not require antigen retrieval. In fact demasking can destroy the epitope after PFA- fixation. Could you please check with your customer again and confirm if the fixation is 4% PFA or formalin fixed (10% NBF – neutral buffered formalin which is 4% formaldehyde)?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Read More

Abcam Scientific Support

Answered on Dec 03 2012

Question

ielen Dank für Ihre Antwort. Kann ich diese Information in einer
Publikation erwähnen?

Mit freundlichen Grüßen

Read More

Abcam community

Verified customer

Asked on Mar 07 2012

Answer

Ja, Sie dürfen die Immunogen-Information gerne in Ihrer Publikation erwähnen.
Ich würde Sie auch gerne bitten die volle Produktnummer (ab32571) anzugeben und uns vielleicht auch eine Kopie Ihrer Publikation zukommen zu lassen. Wir würden uns darüber sehr freuen und würden die Publikation auch auf unser Datenblatt mit aufnehmen.
Ich wünsche Ihnen viel Erfolg mit der Publikation und verbleibe

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Abcam Scientific Support

Answered on Mar 07 2012

Question

Liegt das Immunogen um die Aminosäure 405?

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Abcam community

Verified customer

Asked on Mar 07 2012

Answer

Vielen Dank für Ihren Anruf.
Es freut mich Ihnen nun die Region, in der das Immunogen liegt, genauer angeben zu können:
Aminosäure 1 bis 30 von humanem PYK2.
Ich kann Ihnen somit auch bestätigen, dass Aminosäure 405 nicht im Immunogen enthalten ist.
Ich hoffe, diese Information ist hilfreich und wünsche Ihnen viel Erfolg bei Ihrer Forschung.

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Abcam Scientific Support

Answered on Mar 07 2012

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