Overview

  • Product name

  • Description

    Rabbit polyclonal to ATG9B
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC, ELISAmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic peptide conjugated to KLH from the C-terminus of Human ATG9B

  • Positive control

    • Human placenta tissue, Human spleen tissue

Properties

Applications

Our Abpromise guarantee covers the use of ab117591 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 1 - 2 µg/ml. Predicted molecular weight: 101 kDa.
IHC-P Use a concentration of 5 µg/ml.
ICC Use a concentration of 5 - 10 µg/ml.
ELISA Use at an assay dependent concentration.

Target

  • Function

    Involved in autophagy and cytoplasm to vacuole transport (Cvt) vesicle formation. Plays a key role in the organization of the preautophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle.
  • Tissue specificity

    Highly expressed in placenta (trophoblast cells) and pituitary gland. Not expressed in vascular endothelial.
  • Sequence similarities

    Belongs to the ATG9 family.
  • Cellular localization

    Cytoplasmic vesicle, autophagosome membrane. Under amino acid starvation or rapamycin treatment, redistributes from a juxtanuclear clustered pool to a dispersed peripheral cytosolic pool. The starvation-induced redistribution depends on ULK1 and ATG13.
  • Information by UniProt
  • Database links

  • Alternative names

    • APG9 like 2 antibody
    • APG9-like 2 antibody
    • Apg912 antibody
    • APG9L2 antibody
    • Apgdc2 antibody
    • ATG9 autophagy related 9 homolog B (S. cerevisiae) antibody
    • Atg9b antibody
    • ATG9B_HUMAN antibody
    • Autophagy 9 like 2 protein antibody
    • Autophagy related protein 9B antibody
    • Autophagy-related protein 9B antibody
    • Endothelial nitric oxide synthase antisense antibody
    • eONE antibody
    • Gm574 antibody
    • Nitric oxide synthase 3 antisense antibody
    • Nitric oxide synthase 3 overlapping antisense gene protein antibody
    • Nitric oxide synthase 3-overlapping antisense gene protein antibody
    • NOS3AS antibody
    • Protein sONE antibody
    • SONE antibody
    see all

Images

  • Formalin fixed, paraffin embedded Human placenta tissue labelled with ab117591 at 5 µg/ml
  • Formalin fixed, paraffin embedded Human spleen tissue labelled with ab117591 at 5 µg/ml

References

This product has been referenced in:

See all 2 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mouse Brain and COS7 Monkey Kidney Cells)
Gel Running Conditions
Reduced Denaturing (4-12% gel)
Loading amount
30 µg
Specification
Mouse Brain and COS7 Monkey Kidney Cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 15 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney)
Gel Running Conditions
Reduced Denaturing (4-12% gel)
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 04 2018

Application
Western blot
Sample
African green monkey Cell lysate - whole cell (Kidney)
Gel Running Conditions
Reduced Denaturing (4-12% gel)
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 04 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
African green monkey Cell (Kidney)
Permeabilization
Yes - 0.2% Triron X100
Specification
Kidney
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative
Methanol

Abcam user community

Verified customer

Submitted Sep 27 2017

Question

Dear technical support team: This customer has already purchased ab117591 (Anti-ATG9B antibody), and after he conducted WB with human sample, the result shows wrong band size, therefore he wants to ask for you help to offer any suggestion to him, I also attached the image in this letter, and his experiment step as follow: 1. Order details: Batch number: gr63486-1 Po: xxx Abcam product code: ab117591 Antibody storage conditions (temperature/reconstitution etc) 4℃ 2. Please describe the problem (high background, wrong band size, more bands, no band etc). Wrong band size 3. On what material are you testing the antibody in WB? ·         Species: human ·         What’s cell line or tissue oral cancer cell line ·         Cell extract or Nuclear extract:  cell extract ·         Purified protein or Recombinant protein: purify protein   3.  The lysate How much protein was loaded: 40 ug What lysis buffer was used: lysis buffer from “cell signaling” What protease inhibitors were used: protease cooktail from ”SIGMA” What loading buffer was used: make up by Lab, contain 0.2M Tris-HCl, 10% 2ME, 8% SDS, 40% glycerol and 0.4% Bromophenoblue Phosphatase inhibitors: phosphatise inhibitor from “SIGMA” Did you heat the samples: temperature and time: 95℃ 10mins   4.  Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: Reducing agent: Gel percentage : 10% Transfer conditions: (Type of membrane, Protein transfer verified): NC membrane, 350mA 1hr   5. Blocking conditions Buffer: TBST contain 50mM Tris, 0.85% NaCL and 0.1% tween-20 Blocking agent: milk, BSA, serum, what percentage:5% milk Incubation time:1hr Incubation temperature: room temperture   6. Primary Antibody Species:Rabbit Reacts against: ·         At what dilution(s) have you tested this antibody: 1000X dilute ·         What dilution buffer was used: 0.5% milk in TBST ·         Incubation time: 1hr ·         Incubation temperature: room temperature ·         What washing steps were done: wash 3~4 times/10mins with TBST,   7. Secondary Antibody Species:mouse Reacts against: anti rabbit At what dilution(s) have you tested this antibody: 5000X dilu Incubation time: 1hr Wash steps: wash 3~4 times/10mins with TBST Fluorochrome or enzyme conjugate:HRP Do you know whether the problems you are experiencing come from the secondary?  No  8. Detection method ECl, ECl+, other detection method: ECL 9. Did you apply positive and negative controls along with the samples? Please specify. No 10. Optimization attempts ·         How many times have you tried the Western? 3 times ·         Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):  no ·         Do you obtain the same results every time e.g. are background bands always in the same place?  yes ·         What steps have you altered? 1’ incubation time change from overnight to 1hr, and 2’ Ab diu buffer change from 0.5% milk to 5% milk   Could you please help this customer to solve the problem?

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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 ATG9B-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. Reviewing this case, I would appreciate if you can confirm some further details. Also, I would like to offer some suggestions to help optimise the results from ab117591: 1) May I ask whether you have determined the transcription levels of ATG9B in this cell line, e.g. by RT-PCR? In general, it might be useful to run a positive control alongside the samples, in order to determine the bands that are specific, as the banding pattern obtains might represent unspecific bands. 2) Would you expect the expression of other isoforms of lower molecular weight, as indicated in the literature? 3) I would like to suggest decreasing the boiling temperature to 70ºC and using DTT as reducing agent. Your target is a multi-pass membrane protein, and these proteins have the unfortunate tendency to aggregate during boiling periods with high temperatures and beta- mercaptoethanol due to their hydrophobic nature. 4) Usually, we recommend incubating the primary antibody overnight at +4ºC. The lowered temperature helps to increase the specific signal of the band recognized by the antibody and reduces non-specific background staining. 5) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. I hope some of the suggestions will help to improve the results. As the antibody was purchased in November you are covered by our Abpromise guarantee. Therefore if the suggestions do not improve the results I would be happy to offer a refund or replacement vial. Please do not hesitate to contact me should you have additional questions.

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