Overview

  • Product name
  • Description
    Rabbit polyclonal to PKC gamma
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ELISA, IHC-Fr, WB, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Rabbit, Cow, Drosophila melanogaster
  • Immunogen

    Synthetic peptide:

    DARSPTSPVPVPVM

    , corresponding to amino acids 684-697 of Rat PKC gamma.

  • Positive control
    • recombinant protein

Properties

Applications

Our Abpromise guarantee covers the use of ab4145 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
ELISA Use at an assay dependent concentration.
IHC-Fr 1/250.
WB 1/250. Predicted molecular weight: 84 kDa. using HRP or AP. With a more sensitive detection system, such as enhanced chemiluminescence or ELISA, the antibody may be diluted further.
ICC Use at an assay dependent concentration.

Target

  • Function
    This is a calcium-activated, phospholipid-dependent, serine- and threonine-specific enzyme.
    PKC is activated by diacylglycerol which in turn phosphorylates a range of cellular proteins. PKC also serves as the receptor for phorbol esters, a class of tumor promoters.
  • Tissue specificity
    Expressed in Purkinje cells of the cerebellar cortex.
  • Involvement in disease
    Defects in PRKCG are the cause of spinocerebellar ataxia type 14 (SCA14) [MIM:605361]. Spinocerebellar ataxia is a clinically and genetically heterogeneous group of cerebellar disorders. Patients show progressive incoordination of gait and often poor coordination of hands, speech and eye movements, due to degeneration of the cerebellum with variable involvement of the brainstem and spinal cord. SCA14 is an autosomal dominant cerebellar ataxia (ADCA).
  • Sequence similarities
    Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. PKC subfamily.
    Contains 1 AGC-kinase C-terminal domain.
    Contains 1 C2 domain.
    Contains 2 phorbol-ester/DAG-type zinc fingers.
    Contains 1 protein kinase domain.
  • Information by UniProt
  • Database links
  • Alternative names
    • KPCG_HUMAN antibody
    • MGC57564 antibody
    • OTTHUMP00000067291 antibody
    • PKC-gamma antibody
    • PKCC antibody
    • PKCG antibody
    • PRKCG antibody
    • Protein kinase C gamma antibody
    • Protein kinase C gamma polypeptide antibody
    • Protein kinase C gamma type antibody
    • Protein kinase C, gamma antibody
    • SCA 14 antibody
    • SCA14 antibody
    see all

References

This product has been referenced in:
  • Guo W  et al. Further observations on the behavioral and neural effects of bone marrow stromal cells in rodent pain models. Mol Pain 12:N/A (2016). WB . Read more (PubMed: 27329776) »
  • Rodríguez-Muñoz M  et al. NO-released zinc supports the simultaneous binding of Raf-1 and PKC? cysteine-rich domains to HINT1 protein at the mu-opioid receptor. Antioxid Redox Signal 14:2413-25 (2011). WB ; Mouse . Read more (PubMed: 21235400) »
See all 5 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

Thank you for your technical support and interest.
I would suggest applying longer incubation time (overnight instead of 1 hr). Customer may need to use higher final concentration of this primary antibody and longer permeabilization would be also beneficial.
I hope this helps and if I can assist further, please do not hesitate to contact me.

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Answer

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

I appreciate the time you have spent in the laboratory and your efforts to check the secondary and the expression level in your C6 cells, and I understand your concerns. It is regrettable the results have not been successful.

Indeed, in case there are special protocol conditions required we would mention them on the datasheet. When testing our antibodies, our lab uses 5% BSA as a blocking reagent, as to our experience some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk. However, I agree with you that in this case this might not improve the results. If there is anything to optimise for the blocking conditions, I suggest checking with the manufacturer of the detection system (i.e. Licor/Odyssey), as they recommend not to use milk in some occastions but blocking reagents optimised for their system (http://www.licor.com/bio/products/reagents/odyssey_blocking_buffer/odyssey_blocking_buffer.jsp).

Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results, as your protocol seems fine to me. I can suggest you may have received a bad vial of ab4145, and I apologise for the inconvenience.

Should the attempts you mentioned (regarding the secondary, blocking conditions) not improve the results, I would be pleased to send you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with how you are getting on and how you would like to proceed.

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Question

Good day! We have a customer who has purchased the above product and is having difficulty with this. Please see below for their message :

I am working to optimise our Western Blot for the Abcam PKC-gamma (lot # GR43312). Sadly, we were not able to detect the PKC-gamma in our rat spinal cord lysates. The details and references you’ve provided was helpful. However, I still have a few questions that I hope you’ll be able to help me with so as to figure out why we are not able to detect the PKC-gamma in our rat SC lysates.

I have attached a western blot on the word document.

Western Blot Info:

1) Rat lysate isolated by NP-40 lysis buffer with Triton X-100 substitution the NP-40, supplemented with protease and phosphatise inhibitor cocktails

2) We ran it on a precast NUSep Tris-GLycine SDS-PAGE 4-20% gradient gel

3) We transferred onto PVDF membrane

4) Primary antibodies incubated overnight at 4°C using the PKC-gamma at recommended 1/250 in TBST + 5%milk. Also included a mouse MoAb to beta-tubulin III (as a loading control)

5) Secondary antibodies were from Invitrogen - Alexa Fluor® 680s. Dilutions were done 1/2500 in TBST + 5% milk.

a. Goat Anti-Rabbit IgG (H+L) (to detect PKC antibody) and

b. Alexa Fluor® 680 Anti-mouse IgG (to detect beta-tubulin antibody)

6) Detection was performed by scanning fluorescence in Odyssey.

As you can see from the attached document, the western blot itself has worked well for the tubulin, but not PKC. So my questions are:

1) What is the Abcam recommended protocol including lysis buffer type for isolating tissue lysates to visualise the PKC-gamma? The datasheet just refers to general western blot protocol on your website but does not specify anything specific.

2) The references you listed below for mouse work – it appears they were immunoprecipitating out, and therefore creating a more “concentrated” version of PKC-gamma. Has anyone used this antibody on just crude lysates?

3) Is PKC gamma a membrane or cytosolic protein?

4) Does Abcam have a positive control lysate which you could provide to us that we may run it on a gel to test the efficiency of the PKC antibody we bought?

Any advice would be greatly appreciated. Thank you.

I hope to hear from you soon.

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Answer

Thank you for contacting us.

I am sorry to hear this customer has been experiencing problems with one of our PKC gamma antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints. I appreciate the time taken to submit further information to us, and after reading the answers provided I would like to ask some additional questions:

1) Could you please confirm how the secondary goat anti-rabbit IgG (H+L) antibody is labeled? Is it also Alexa Fluor 680, or would you detect this antibody with chemiluminescence (HRP+substrate)? Also, I would appreciate if you could confirm that this antibody is working (e.g. when using it with other primaries).

2) How was the membrane blocked? And have you tried to incubate the primary antibody in dilution buffer with less or even without blocking agent? If high background is not an issue, some antibodies produce a much stronger signal if diluted in buffer with low concentrations (0.5 – 0.25%) of milk or BSA, or none at all.

Regarding your questions:
1) The protocol and lysis buffer depends on the tissue type and any other targets you might want to detect simultaneously. This is why we refer to general Western blot protocols (attached). Having said this though, I am happy to let you know that the NP40 lysis buffer you used seems absolutely fine for detecting the PKC gamma, so I am confident that this is not the cause of the problem.

2) The references listed for ab4145 are only the references we know of that cited this particular antibody, although there might be more available. Please be reassured that this PKC gamma antibody has been tested in our labs and we guarantee it to work in Western blot with rat samples of any source that contains the protein, be it crude or concentrated lysate.

3) PKC gamma is a cytosolic protein, and you may find more detailed information in databases such as PubMed or SwissProt (http://www.uniprot.org/uniprot/P63319) or in our Pathway Posters (Cellular Apoptosis, attached).

4) Unfortunately, we currently do not have a positive control commercially available for you to test the antibody. I would recommend to check this antibody with a positive sample such as mouse or rat brain tissue lysate (I may assume that you can get hands on this tissue as you are working with rat spinal cords?) or even HeLa or A431 cell lysates which are widely distributed and you might get a small sample from a neighboring lab.

Please be reassured that you are covered by our Abpromise and if it turns out that the antibody is not working although the conditions are right (detection of the secondary antibody, blocking reagents etc), we will offer you a free of charge replacement or a credit note.

I look forward to receiving your reply.

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Answer

Thank you for your e-mail. It is good to know that the customer uses a good lysis buffer, secondary antibody and detection system. However, the form you have sent us states the customers used NRK,NR8383, SMC and HSC-T6 cell lines, which are not neuronal. Can the customer please clarify what neuronal cell lines he has tried and detail the evidence that those cells should express the protein (clarify by sending a reference)? Indeed, even cultured neonatal rat brain cells (cerebellar granule cells) do not express high levels of the protein according to the following reference : TCDD alters PKC signaling pathways in developing neuronal cells in culture. Kim SY, Lee HG, Choi EJ, Park KY, Yang JH. Chemosphere. 2007 Apr;67(9):S421-7. My primary concern is therefore that the customer's cells do not express high levels of PKC gamma. The laboratory has tested the antibody on recombinant protein; if the customer would like to access this positive control please let me know and I will try to see if we can add this to our catalogue.

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Answer

I can confirm that we recommend a positive control of recombinant protein or as PKC gamma is expressed solely in the brain and spinal cord and its localization is restricted to neurons to try brain or spinal cord lysate, my apologies for the delay in confirming this information. The customer has not detailed the lysis buffer used, I would recommend to make sure it is strong enough, such as NP40 or RIPA buffer, to enable adequate extraction of the protein. It would also be worth using an ECL+ or Pierce supersignal kit which are more sensitive than Ecl, and to test that the secondary antibody is not damaged and responsible for the lack of signal.

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. Although ab4145 is predicted to react with mouse, it has not yet been tested so we are unable to guarantee results. The antibody is guaranteed to work with rat tissue and I would suggest trying this as a positive control. I would also suggest incubating with the primary for overnight at 4C and checking to see that the secondary antibody is working properly. Also, decrease the blocking time and if you haven't already, increase the detection efficiency by using signal amplification systems such as ABC. I hope this helps. Please don't hesitate to contact us again if you need additional assistance.

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Answer

Thank you for your patience and for the details that you have provided. What was the lot number (located on the vial) that you received? What was the Abcam order number or purchase order number that was used? We haven't had any other complaints but I want to check with the originator of the antibody to see if they have had any complaints about the lot and if they have any suggestions.

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