Overview

  • Product name
  • Description
    Rabbit polyclonal to PDE5A
  • Host species
    Rabbit
  • Specificity
    This antibody detects 99 and 89 kDa PDE5A proteins in various tissues.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human PDE5A. Synthetic peptide common to all PDE5A variants, taken within amino acid region 120-170 of human PDE5A protein.
    (Peptide available as ab150691)

  • Positive control
    • Rat lung homogenate.
  • General notes
    Storage of very dilute antibody solutions is not recommended.

Properties

Applications

Our Abpromise guarantee covers the use of ab14672 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-P 1/10. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 µg/ml.
WB 1/500. Detects a band of approximately 89, 99 kDa (predicted molecular weight: 105 kDa).Can be blocked with Human PDE5A peptide (ab150691).
IP 1/250.

Target

  • Function
    Plays a role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. This phosphodiesterase catalyzes the specific hydrolysis of cGMP to 5'-GMP.
  • Tissue specificity
    Expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung.
  • Pathway
    Purine metabolism; 3',5'-cyclic GMP degradation; GMP from 3',5'-cyclic GMP: step 1/1.
  • Sequence similarities
    Belongs to the cyclic nucleotide phosphodiesterase family.
    Contains 2 GAF domains.
  • Domain
    Composed of a C-terminal catalytic domain containing two putative divalent metal sites and an N-terminal regulatory domain which contains two homologous allosteric cGMP-binding regions, A and B.
  • Post-translational
    modifications
    Phosphorylation is regulated by binding of cGMP to the two allosteric sites.
  • Information by UniProt
  • Database links
  • Alternative names
    • 5''-cyclic phosphodiesterase antibody
    • CGB PDE antibody
    • CGB-PDE antibody
    • CGBPDE antibody
    • cGMP binding cGMP specific 3' 5' cyclic nucleotide phosphodiesterase antibody
    • cGMP binding cGMP specific 35 cyclic nucleotide phosphodiesterase antibody
    • cGMP binding cGMP specific phosphodiesterase antibody
    • cGMP binding/cGMP specific phosphodiesterase antibody
    • cGMP specific 3'5' cyclic phosphodiesterase antibody
    • cGMP specific phosphodiesterase PDE5A2 antibody
    • cGMP specific phosphodiesterase type 5A antibody
    • cGMP-binding cGMP-specific phosphodiesterase antibody
    • cGMP-specific 3'' antibody
    • CN5A antibody
    • CN5N antibody
    • PDE 5 antibody
    • PDE 5A antibody
    • PDE5 antibody
    • Pde5a antibody
    • PDE5A_HUMAN antibody
    • PDE5A1 antibody
    • Phosphodiesterase 5A antibody
    • Phosphodiesterase 5A cGMP specific antibody
    • Phosphodiesterase isozyme 5 antibody
    see all

Images

  • ab14672 (4µg/ml) staining PDE5A in human oesophagus using an automated system (DAKO Autostainer Plus). Using this protocol there is staining of the cytoplasm and nuclei in stratified squamous epithelium cells.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • ICC/IF image of ab14672 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14672, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Zhang Q  et al. PPAR? Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling. PPAR Res 2018:6970407 (2018). Read more (PubMed: 29686697) »
  • Yan L  et al. Icariside II Effectively Reduces Spatial Learning and Memory Impairments in Alzheimer's Disease Model Mice Targeting Beta-Amyloid Production. Front Pharmacol 8:106 (2017). Read more (PubMed: 28337142) »
See all 6 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Te pido disculpas por la espera.
El péptido usado como inmunógeno para generar ab14672 se ha añadido finalmente al catálogo.
El número de catálogo del péptido es ab150691.
Espero que con ayuda del péptido consigáis demostrar la especificidad del anticuerpo.
Te recuerdo que todos nuestros productos tienen 6 meses de garantía, y si tuvieras algún problema al utilizarlos, te animo a que nos contactes.

Read More

Question

Dear Technical Team,

Thank you for your mail.

Please find below the reply from the customer & also arrange for the needful to update the customer accordingly on it.

Looking forward towards your valued response on it.

With Best Regards,


Dear Sir,

Yes, the same secondary antibody is being used for all these antibodies, even being used with primary antibodyof other brands, working well.

Please replace these antibodies.

regards,














----- Original Message -----

From: mailto:technical@abcam.com

To: mailto:nulife@bol.net.in

Sent: Wednesday, January 25, 2012 3:56 PM

Subject: Reply from Abcam to your enquiry regarding ab14672, ab60532, ab71700, ab81505 [CCE3453323]



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https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header









Dear Nadeem



We have an answer to your inquiry:




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 enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. I would like to reassure you that these antibodiesare tested and covered by our 6 month guarantee forWB. All the antibodies, except for ab81505Arg2 antibody,are guaranteed for rat samples.



Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details:



1. Please confirm the Abcam order reference number and date of purchase for each of these antibodies.



2. Could you confirm the same secondary antibody is being used for allof these antibodies? Is the current vial of secondary antibody working well with any other primary antibodies? Please provide further details of the secondary (supplier and catalog number).



3. I can recommend to try somefresh samples.



4. I can suggest to include a loading control such as GAPDH in order to assess the quality of the sample and the transfer to the membrane.



5. I can recommend to include some positive controls if they have not already been tried. Reviewing the information on the datasheets, anddetails fromthe SwissProt/Uniprot protein database links, I can suggest for example:



PDE5A (ab 14672)

Rat lung homogenate should already be a suitable control



cGK1 (ab 60532)

Epithelial cells of thymus, dendritic cells of lymph node, and in the basal cell layer of epithelia such as epidermis, nasopharynx, respiratory tract, salivary gland, and esophagus express cGK1,



Sphk1 (ab 71700)

I am sorry I am not able to confirm what expression level isexpected in lung? Do you have any further information? I can suggest adult liver, kidney, heartor skeletal muscle would be a suitable control.



4. Arg2 (ab 81505)

I am sorry I am not able to confirm whatexpression level is expected in the lung? Do you have any further information? This protein is expressed most strongly in kidney and prostate,

I can recommend to try a humansample positive control as human samlples are tested and covered by the guarantee. Rat samples arepredicted only due to high sequence alignment, I am sorry they have not been tested.



In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.



I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.





Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3453323




Your original inquiry to Abcam:





Dear Technical Team,







Hope this e-mail finds you well!








Kindly look into the belowmail from one this customer & advise us on it, enabling us to update him accordingly.







Looking forward towards your valued response on it.







With Best Regards,










Dear Sir,







We procured the following antibodies from you via supply order : 201/DIPAS

1. PDE5A (ab 14672)

2. cGK1 (ab 60532)

3. Sphk1 (ab 71700)

4. Arg2 (ab 81505)



we have been testing these antibodies for the past 3 months and their functionality is now being doubted as we have loaded increasing

amounts of protein sample during western blot experiments (starting with 30ug - till 50 ug) and tried extended primary incubations

(3hr/5hr/overnight) at the recommended antibody concentrations (1:1000-1:500), but there seems to be no binding of the primary

antibody to the blots as observed on developing through ECL detection system (SIGMA).This hasseriouslyaffectedprogressof our research project.





kindly take notice of the problem and if possible try to replace the above, as the above purchases are still in the warranty period.







Enclosed please find the required information for further action.





regards,


















Order Details

Antibody code :

Problem :

- No Signal or Weak Signal

Lot number :

Purchase order number : 201/DIPAS/MMG/DIP -251/111/11-12/A2.3

or preferably Abcam order number





General Information

Antibody storage conditions (temperature/reconstitution etc)

Stored at -20°C in aliquots and working dilutions stored at 4°C.





Description of the problem (high background, wrong band size, more bands, no band etc.)

No bands



Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)

Rat whole lung homogenate prepared in RIPA lysis buffer



Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

RIPA lysis buffer fortified with 1mM PMSF and protease inhibitor cocktail (Roche). Sample was appropriately diluted with 6X Laemelli buffer and water and boiled for 10 minutes in a water bath.



Amount of protein loaded

Started with 30μg and increased the protein loaded/well till 50 μg

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)

Reducing gel (SDS-PAGE) , 5% stacking and 10% resolving. Gel was run at 60V for 4 hours after stacking at 55V.

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)

Transfer : 15V at room temperature for 1hr in a semi-dry blotting apparatus.

Transfer buffer: 0.025M Tris base, 0.192M Glycine, 20% Methanol (pH 8.3)

Blocking Buffer: 5% BSA in TBST

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

PDE5A (ab14672), Sphk1(ab 71700), Arg 2( ab81505) : all manufactured by Abcam

Species reactivity : Human, Mouse, Rat

Origin: Rabbit

Dilutions: 1:500

Diluent: TBST+ 5% BSA

Incubation time: 3 hours – overnight (at 37°C / 4°C, as suitable)

Washing: 3-5 washings for 10 minutes with TBST at room temperature

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

Anti-Rabbit IgG (Santa Cruz)

Origin: Goat

Dilution: 1:5000

Diluent: TBST+5%BSA

Incubation time: 2-3 hours at 37°C

Washing: 3-5 washings for 10 minutes with TBST at room temperature

Detection method (ECL, ECLPlus etc.)

Detection Method: ECL (SIGMA) + Kodak Blue Sensitive X-Ray Film+ Kodak Dental developer/fixer



Positive and negative controls used (please specify)

Positive control: Blots probed with above specifies antibodies were stripped, blocked and re-probed for β-actin. Specific bands of β-actin were obtained on the exposed film.



Optimization attempts (problem solving)

How many times have you tried the Western?



9 times for each antibody



Have you run a "No Primary" control? Yes No Not required



Do you obtain the same results every time? Yes No

e.g. are the background bands always in the same place?

No, only blank x-ray film was obtained each time





What steps have you altered?

- We have increased the protein concentration loaded/well from 30ug to 50ug.

- Used the recommended antibody dilutions at high concentration and longer incubation periods – 3 hours to overnight.

- The transfer efficiency was ensured by ponceau staining.

Additional Notes

Attached images are representative of each antibody probing procedure (only the highest concentration of protein loaded with longest primary antibody incubation has been depicted in the images)












Best regards,
Kate

Kate Hayes
Scientific Support Supervisor
Abcam plc
www.abcam.com



Abcam Customer Services and Scientific Support Team
https://www.abcam.com/index.html?pageconfig=technical&utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Body

[CCE3453323]










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Answer

Thank you for providing this further information.

I appreciate your cooperation and understand your concerns. It is regrettable the results have not been succesful.

In order to help with our invesigation of this case before deciding how to proceed, I would appreciate if you are able to provide more detail as requested in the previous email. This information will also be helpful to our quality monitoring.

1. Please confirm the Abcam order reference number and date of purchase for each of these antibodies.

2. I can suggest to include a loading control such as GAPDH in order to assess the quality of the sample and the transfer to the membrane.
Could you confirm the results from the loading control?

3. I can recommend to include some positive controls if they have not already been tried.

Please confirm what level of expression is expected in the samples. Which positive controls have been tried for each antibody?

Particularly for ab81505 which has not been tested and guaranteed in rat, please confirm if a human positive control has been tried as suggested.

Thank you for your time. I look forward to hearing from you with the requested information and hope we can resolve this case as soon as possible.

Read More

Question

Dear Technical Team,

Hope this e-mail finds you well!

Kindly look into the belowmail from one this customer & advise us on it, enabling us to update him accordingly.

Looking forward towards your valued response on it.

With Best Regards,


Dear Sir,

We procured the following antibodies from you via supply order : 201/DIPAS
1. PDE5A (ab 14672)
2. cGK1 (ab 60532)
3. Sphk1 (ab 71700)
4. Arg2 (ab 81505)

we have been testing these antibodies for the past 3 months and their functionality is now being doubted as we have loaded increasing
amounts of protein sample during western blot experiments (starting with 30ug - till 50 ug) and tried extended primary incubations
(3hr/5hr/overnight) at the recommended antibody concentrations (1:1000-1:500), but there seems to be no binding of the primary
antibody to the blots as observed on developing through ECL detection system (SIGMA).This hasseriouslyaffectedprogressof our research project.

kindly take notice of the problem and if possible try to replace the above, as the above purchases are still in the warranty period.

Enclosed please find the required information for further action.

regards,






Order Details
Antibody code :
Problem :
- No Signal or Weak Signal
Lot number :
Purchase order number : 201/DIPAS/MMG/DIP -251/111/11-12/A2.3
or preferably Abcam order number

General Information
Antibody storage conditions (temperature/reconstitution etc)
Stored at -20°C in aliquots and working dilutions stored at 4°C.

Description of the problem (high background, wrong band size, more bands, no band etc.)
No bands
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Rat whole lung homogenate prepared in RIPA lysis buffer
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
RIPA lysis buffer fortified with 1mM PMSF and protease inhibitor cocktail (Roche). Sample was appropriately diluted with 6X Laemelli buffer and water and boiled for 10 minutes in a water bath.
Amount of protein loaded
Started with 30μg and increased the protein loaded/well till 50 μg
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Reducing gel (SDS-PAGE) , 5% stacking and 10% resolving. Gel was run at 60V for 4 hours after stacking at 55V.
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Transfer : 15V at room temperature for 1hr in a semi-dry blotting apparatus.
Transfer buffer: 0.025M Tris base, 0.192M Glycine, 20% Methanol (pH 8.3)
Blocking Buffer: 5% BSA in TBST
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
PDE5A (ab14672), Sphk1(ab 71700), Arg 2( ab81505) : all manufactured by Abcam
Species reactivity : Human, Mouse, Rat
Origin: Rabbit
Dilutions: 1:500
Diluent: TBST+ 5% BSA
Incubation time: 3 hours – overnight (at 37°C / 4°C, as suitable)
Washing: 3-5 washings for 10 minutes with TBST at room temperature
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Anti-Rabbit IgG (Santa Cruz)
Origin: Goat
Dilution: 1:5000
Diluent: TBST+5%BSA
Incubation time: 2-3 hours at 37°C
Washing: 3-5 washings for 10 minutes with TBST at room temperature
Detection method (ECL, ECLPlus etc.)
Detection Method: ECL (SIGMA) + Kodak Blue Sensitive X-Ray Film+ Kodak Dental developer/fixer
Positive and negative controls used (please specify)
Positive control: Blots probed with above specifies antibodies were stripped, blocked and re-probed for β-actin. Specific bands of β-actin were obtained on the exposed film.
Optimization attempts (problem solving)
How many times have you tried the Western?
9 times for each antibody
Have you run a "No Primary" control? Yes No Not required
Do you obtain the same results every time? Yes No
e.g. are the background bands always in the same place?
No, only blank x-ray film was obtained each time

What steps have you altered?
- We have increased the protein concentration loaded/well from 30ug to 50ug.
- Used the recommended antibody dilutions at high concentration and longer incubation periods – 3 hours to overnight.
- The transfer efficiency was ensured by ponceau staining.
Additional Notes
Attached images are representative of each antibody probing procedure (only the highest concentration of protein loaded with longest primary antibody incubation has been depicted in the images)

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 enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. I would like to reassure you that these antibodiesare tested and covered by our 6 month guarantee forWB. All the antibodies, except for ab81505Arg2 antibody,are guaranteed for rat samples.

Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details:

1. Please confirm the Abcam order reference number and date of purchase for each of these antibodies.

2. Could you confirm the same secondary antibody is being used for allof these antibodies? Is the current vial of secondary antibody working well with any other primary antibodies? Please provide further details of the secondary (supplier and catalog number).

3. I can recommend to try somefresh samples.

4. I can suggest to include a loading control such as GAPDH in order to assess the quality of the sample and the transfer to the membrane.

5. I can recommend to include some positive controls if they have not already been tried. Reviewing the information on the datasheets, anddetails fromthe SwissProt/Uniprot protein database links, I can suggest for example:

PDE5A (ab 14672)
Rat lung homogenate should already be a suitable control

cGK1 (ab 60532)
Epithelial cells of thymus, dendritic cells of lymph node, and in the basal cell layer of epithelia such as epidermis, nasopharynx, respiratory tract, salivary gland, and esophagus express cGK1,

Sphk1 (ab 71700)
I am sorry I am not able to confirm what expression level isexpected in lung? Do you have any further information? I can suggest adult liver, kidney, heartor skeletal muscle would be a suitable control.

4. Arg2 (ab 81505)
I am sorry I am not able to confirm whatexpression level is expected in the lung? Do you have any further information? This protein is expressed most strongly in kidney and prostate,

I can recommend to try a humansample positive control as human samlplese are tested and covered by the guarantee. Rat samples arepredicted only due to high sequence alignment, I am sorry they have not been tested.

In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Thank you for your enquiry. With necrotic tissue, it is very likely that the proteins are already being broken down and degraded by the enzymes that are already present. I am sorry it would be very difficult to say and guarantee how much Endothelin 1 or PDE5A would still be in the sample for detection by the antibody. If the proteins are released from the cell during necrosis, the localization of the protein may also be affected.  If the tissue is already necrotic before fixation, then there are no fixation methods that would specifically help in this case. I can suggest to try the usual fixation methods. I can suggest it would be important to consider including positive control tissue. For example: PDE5A antibody (ab14672) has been used successfully in rat human heart tissue. We do have the following slides available: ab4332  Heart (human): aortic valve normal tissue slides Applications: IHC-P, ISH   Endothelin 1 antibody (ab2786) has been used successfully in human bowel. We do have the following slides available: ab4327 Colon (human) normal tissue slides Applications: IHC-P, ISH    I hope this information will be helpful to you.  If you have any further questions, please do not hesitate to contact me.    

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (right ventricle)
Specification
right ventricle
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Mar 12 2008

Answer

Thank you for your enquiry. At the moment, ab14672 is the only PDE5 antibody that we have. Ab14672 has been tested for application in Western blotting and IP and tested for cross-reactivity with Human, Mouse and Rat. If you decide to go ahead and purchase this product, please let us know how you get on and in return we will award you 50 points with the Abcam Loyalty Scheme which can be redeemed on a number of rewards. To see if another company may have what you are looking for, you can click on the link to "The World's Antibody Gateway". The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogues of all online antibody companies (currently 249). If you have any additional questions, please contact us again.

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