Overview

  • Product name
    Anti-Chromogranin B antibody
    See all Chromogranin B primary antibodies
  • Description
    Rabbit polyclonal to Chromogranin B
  • Host species
    Rabbit
  • Specificity
    Ab12242 recognises chromogranin B.
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-Fr, ICC, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    C-terminal 15 amino acids of chromogranin B conjugated to ovalbumin.

Properties

Applications

Our Abpromise guarantee covers the use of ab12242 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 1/100 - 1/500. Predicted molecular weight: 78.2 kDa.
ICC/IF Use at an assay dependent concentration. PubMed: 24454876
IHC-Fr 1/100 - 1/500.
ICC Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.

Target

  • Function
    Secretogranin-1 is a neuroendocrine secretory granule protein, which may be the precursor for other biologically active peptides.
  • Tissue specificity
    Expressed in the adrenal medulla, and in pheochromocytoma. Not expressed in liver.
  • Sequence similarities
    Belongs to the chromogranin/secretogranin protein family.
  • Post-translational
    modifications
    Extensively processed by limited proteolysis at conserved basic residues. Alternative processing are seen in different tissues.
  • Cellular localization
    Secreted. Neuroendocrine and endocrine secretory granules.
  • Information by UniProt
  • Database links
  • Alternative names
    • CCB peptide antibody
    • Cg B antibody
    • CgB antibody
    • Chgb antibody
    • Chromogranin-B antibody
    • ChromograninB antibody
    • SCG1 antibody
    • SCG1_HUMAN antibody
    • Secretogranin 1 antibody
    • Secretogranin B antibody
    • Secretogranin I antibody
    • SgI antibody
    see all

Images

  • ab12242(1:1000) staining Chromogranin B in human pancreas (IgG inset) using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of Chromogranin B in secretory compartments of the pancreas.
    Insert panel depicts negative control (no primary antibody).
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH9.0 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.

References

This product has been referenced in:
  • Stephens SB  et al. The Prohormone VGF Regulates ß Cell Function via Insulin Secretory Granule Biogenesis. Cell Rep 20:2480-2489 (2017). WB, ICC/IF . Read more (PubMed: 28877479) »
  • Zhu W  et al. Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy. J Clin Invest 127:4569-4582 (2017). Read more (PubMed: 29058688) »
See all 7 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Question

1) Abcam product code: ab12242
2) Abcam order reference number or product batch number: GR-26159-2
3) Description of the problem: (1) Abnormally high background for the
Western blot and (2) expected band at ˜80 kDa was also detected in lysates
of non-secretory cells (refer to attached figure)
4) Sample preparation:
Type of sample: Whole cell lysates
Lysis buffer: 1x SDS sample buffer
Protease inhibitors: No
Phosphatase inhibitors: No
Reducing agent: 2-mercaptoethanol
Boiling for ≥5 min? Yes
Protein loaded ug/lane or cells/lane: > 6 ug/lane
Positive control: PC12 cells
Negative control: non-secretory cells
5) Percentage of gel: 10%
Type of membrane: PVDF
Protein transfer verified: Yes
Blocking agent and concentration: 0.5% milk powder in TBS
Blocking time: overnight
Blocking temperature: 4oC
6) Primary antibody: ab12242
Concentration or dilution: 1.0 μg/ml
Diluent buffer: TBS with 0.5% milk powder
Incubation time: 2 h
Incubation temperature: Ambient temperature
7) Secondary antibody:
Species: Sheep
Reacts against: Rabbit IgG
Concentration or dilution: 1:10000
Diluent buffer: TBS with 0.5% milk powder
Incubation time: 1 h
Incubation temperature: Ambient temperature
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer: TBS with 0.5% milk powder
Number of washes: 4 times
9)Detection method: Enhanced chemiluminescence
10) How many times have you run this staining? One
Do you obtain the same results every time?
What steps have you altered to try and optimize the use of this antibody?

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 this antibody is tested and covered by our guarantee for WB.


Reviewing this case, I would like to offer some suggestions to help optimise the results from ab12242.

1.) Chromogranin B is only predicted to be of the size of 78kDa. It is highly phosphorylated on multiple sites which affects its apparent molecular weight on a gel. The lab confirmed that they see this protein at a size of ca 110kDa on gels due to all the modifications.
On the image send to me there is a band at the right size (ca117kDa) that appears only in the secretory rat cells.

Therefore I believe this is a case of a high background and would like to offer the following recommendation for the optimization:

1.) I suggest to use proteinase inhibitors. Since all bands are smaller than the expected protein, it could be degradation products.

2.) I also recommend to run a no primary control to verify that the secondary alone is not producing any bands.

3.) Using BSA as blocking agent can lead to much better results with some antibodies. This has to be tested experimentally, since it is not known why some antibodies work better with BSA.

4.) I suggest to incubate the primary antibody over night at 4C. This ensures the most specific staining.

5.) If antibodies are used to concentrated they start binding un-specifically. I therefore suggest to further dilute the primary antibody.


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 email. We unfortunately do not have ab8205 anymore and we also do not have exactly same product in catalogue.  However we do have many "Chromogranin A" antibodies that are very well characterized. You may consider trying.  The catalogue number of few of these is; ab43844 ab45179 ab79276 ab80787 ab75939 These antibodies are characterized with rat samples and are fully guaranteed to work. I am sorry to hear that you had problem with ab12242 it should have worked in IHC-P. Have you contacted scientific support team and discussed the problem. Under the Abpromise scheme you could have received a free of charge replacement product. Click below for Abpromise guarantee; https://www.abcam.com/index.html?pageconfig=abpromise I hope this information will help for ordering these products. Should you have any question 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
Western blot
Sample
Human Tissue lysate - other (Cerebrospinal fluid (CSF))
Loading amount
3 µg
Specification
Cerebrospinal fluid (CSF)
Gel Running Conditions
Non-reduced Denaturing (12% SDS)
Blocking step
Milk as blocking agent for 17 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Ms. Christiane Rewerts

Verified customer

Submitted May 29 2009

Answer

Thank you for your enquiry and your interest in our products. Here is the protocol that was used to develop this antibody: Frozen pellets of PC12 pheochromocytoma cells were thawed (1 g wet weight/10 ml) in a buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM EDTA, 5 mM 2-mercaptoethanol, and 0.1 mM PMSF at 4 C. The suspension was sonicated on ice using five 10-sec bursts at a setting of 5 with a Branson Sonifier 250 and centrifuged at 100,000 x g for 30 min at 4 C. The supernatant was adjusted to 0.1 M NaCl, boiled for 5 min, and centrifuged again at 100,000 x g for 30 min. The resulting supernatant was concentrated 10-fold using an Amicon Ultrafiltration Cell and a Diaflo YM 30 Ultrafilter (30,000 mol wt cut-off). The protein concentration for the resulting fraction was determined by the method of Lowry et al. using BSA as the standard. Supernatants were boiled in an equal volume of 2-fold electrophoresis sample buffer containing 2% SDS, 0.004% bromphenol blue, 20% glycerol, 4% beta-mercaptoethanol, and 0.12 M Tris-HCl, pH 6.7. Analysis was performed by SDS-PAGE on 10% or 12% gels followed by Western blotting with the antibody at a dilution of 1:100. We hope this information will be useful for you.

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Question
Answer

This product is sold as whole antiserum, not as purified antibody. In general, we estimate that the total Ig concentration in serum is approx. 10 mg/ml, and the specific antibody component is approx. 10% of this. I hope this helps.

Read More

Answer

Thank you for your enquiry. This product is whole, undiluted antiserum, so it is not in a storage buffer. To dilute for use, we recommend either PBS or a buffer/medium that is compatible with the user's assay system.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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