Overview

  • Product name
    Anti-PKA alpha + beta (catalytic subunits) (phospho T197) antibody
    See all PKA alpha + beta (catalytic subunits) primary antibodies
  • Description
    Rabbit polyclonal to PKA alpha + beta (catalytic subunits) (phospho T197)
  • Host species
    Rabbit
  • Specificity
    This antibody exibited a preference for PKA catalytic subunit beta in some tested cell lines.
  • Tested applications
    Suitable for: WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Cow, Pig
  • Immunogen

    Synthetic phosphopeptide derived from a region of human PKA that contains threonine 197.

  • Positive control
    • Forskolin-treated NIH3T3 cells, and Y-1 mouse adrenal cortical cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab5815 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 Use a concentration of 0.1 - 0.75 µg/ml. Detects a band of approximately 42 kDa.

Target

  • Relevance
    PRKACA and PRKACB are members of the Ser/Thr protein kinase family and are a catalytic subunit of cAMP-dependent protein kinase. cAMP is a signaling molecule important for a variety of cellular functions. cAMP exerts its effects by activating the cAMP-dependent protein kinase, which transduces the signal through phosphorylation of different target proteins. The inactive kinase holoenzyme is a tetramer composed of two regulatory and two catalytic subunits. cAMP causes the dissociation of the inactive holoenzyme into a dimer of regulatory subunits bound to four cAMP and two free monomeric catalytic subunits.
  • Cellular localization
    Cytoplasm. Nucleus. Note=Translocates into the nucleus (monomeric catalytic subunit). The inactive holoenzyme is found in the cytoplasm
  • Database links
  • Alternative names
    • cAMP dependent protein kinase beta catalytic subunit antibody
    • cAMP dependent protein kinase alpha catalytic subunit antibody
    • cAMP dependent protein kinase catalytic subunit alpha antibody
    • cAMP dependent protein kinase catalytic subunit beta antibody
    • PKA C alpha antibody
    • PKA C beta antibody
    • PKACA antibody
    • PKACB antibody
    • PRKACA antibody
    • PRKACB antibody
    • Protein kinase cAMP dependent catalytic alpha antibody
    • Protein kinase cAMP dependent catalytic beta antibody
    see all

Images

  • Peptide Competition and Phosphatase Treatment: Lysates prepared from Y1 Adrenocortical cells were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda  phosphatase (5), blocked with a 5% BSA-TBST buffer for two hours at room temperature, then incubated with 0.35 µg/mL ab5815 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG HRP conjugate  and bands were detected using the Pierce SuperSignalTM method. The data show that the peptide corresponding to PKA [pT197] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

    Peptide Competition and Phosphatase Treatment: Lysates prepared from Y1 Adrenocortical cells were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer for two hours at room temperature, then incubated with 0.35 µg/mL ab5815 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that the peptide corresponding to PKA [pT197] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

References

This product has been referenced in:
  • Jiang Z  et al. Blocking mammalian target of rapamycin alleviates bone cancer pain and morphine tolerance via µ-opioid receptor. Int J Cancer 138:2013-20 (2016). WB ; Rat . Read more (PubMed: 26566757) »
  • Panas MW  et al. Trace amine associated receptor 1 signaling in activated lymphocytes. J Neuroimmune Pharmacol 7:866-76 (2012). Human . Read more (PubMed: 22038157) »
See all 3 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

BATCH NUMBER 111494 ORDER NUMBER Forgot DESCRIPTION OF THE PROBLEM No signal or weak signal: No signal at all in cultured cell immunostaining; No signal at all in Western blot against whole cell lysis SAMPLE SH-SY5Y cells and HEK293 cell cultured on 2-well slides, treated/untreated with Ethanol; HEK293 cell lysis treated/untreated with Ethanol, runned on 12%SDS gel and transfered to PVDF membrane. PRIMARY ANTIBODY Abcam Ab5815/Rabbit/2.5% BSA in TBS/1:200 or 1:500 or 1:1000/4oC overnight/0.1% TBST 15 minutes DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED PKA transfected/non-transfected HEK293 cells work as positive or negative control, and each has 100mM Ethanol treated and untreated samples. According to references, ethanol treatment increases PKA phosphralation. And even if ethanol doesn't increase PKA phosphralation in this case, there still should be phosphrelated PKA in normal condition HEK293 cell lysis. In other words, there should always be specific phosphrelated PKA bands in the cell lysis, but unfortunately it never appears,not even any non-specific bands, after lots of repeats and various dilutions. ANTIBODY STORAGE CONDITIONS -20oC SAMPLE PREPARATION Western: M-PER lysis buffer(PIERCE)/Protease Inhibitor Tablet(Roche)/1XLDS loading buffer(Invitrogen), 100oC 5minutes for denature For western, 1:200~1000 dilution of Ab5815 in 2.5%BSA incubate overnight, other procedures are standard Immunostaining:4%PFA fixed cells grown on 2-well slides(Clontech) 15 minutes,0.1% TritonX-100 penetrance 15minutes,5%BSA blocked 1 hour, 1:100 or 1:250 dilution of Ab5815 in 2.5% BSA overnight incubation. Other procedure are standard. AMOUNT OF PROTEIN LOADED 20~30 ug/lane ELECTROPHORESIS/GEL CONDITIONS 12% SDS reducing gel, 100V 2 hours TRANSFER AND BLOCKING CONDITIONS Standard transfer buffer (Invitrogen) and blocking buffer (5% fat-free milk in TBS) SECONDARY ANTIBODY Santa Cruz Inc/Goat against Rabbit HRP-conjugated 2nd Antibody/2.5% fat-free milk/1:5000/RT 1 hour/3 times wash, 0.1% TBST 15 minutes HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Dilution, 1:200; 1:500; 1:1000. ADDITIONAL NOTES I believe this batch of antibody Ab5815 is a bad product, the antibody died when it arrived. When it arrived last year, I first tried it in immunostaining with 1:100/1:250 dilution but never get any signal, I thought it may not suitable for immunostaing. Then in April when I use it for western blots, it never gave any, literally, any bands on the blots. I changed the dilution from 1:1000 to 1:500 to 1:200 but never got anything. This made me believe this batch of Ab5815 was dead when it was first packaged. Because it arrived in blue ice pretty fast after ordering and had been kept in -20oC ever since, so I think it's Abcam's package problem or this batch wasn't good. I have used a lot of Abcam antibodies and all others worked fine except this one was so frustrating, I reqest a free replacement for this antibody as soon as possible. Best Yiguo

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and you have tried many of the suggestions that I would recommend. You have tried various dilutions and induced PKA alpha phosphorylation. Quality is important to Abcam and therefore I would like to offer you a credit note against your original purchase provided that the antibody was purchased within the past 90 days. If this is the case please email me details of the order including the order number and date of purchase. I look forward to hearing from you.

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Answer

Thank you for your enquiry regarding an antiPKA which is specific for PKA catalytic subunit(both the alpha and beta forms)for use in Western blot. The product catalogue number which matches your querie specifications is ab5815. Please refer to the online product datasheet for detailed information such as pricing and technical data for this antibody. If you need additional information to that provided on the datasheet, please contact us again and we will gladly assist you.

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Answer

I'm afraid I don't know. ab8356 was shown to work in IF by one of our customers, Dr Martha Simpson-Holley . Your customer may contact her via the e-mail link in the reviews section of Abcam's online datasheet to request more details of her protocol. To our knowledge, ab5815 has only been tested in WB, so I am unable to advise how tissue samples should be prepared when it's used in IHC.

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