Overview

  • Product name

  • Description

    Mouse polyclonal to AFG3L2
  • Host species

    Mouse
  • Tested applications

    Suitable for: ICC/IF, WBmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Full length protein (Human)

  • Positive control

    • AFG3L2 transfected 293T cell lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab68023 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
ICC/IF Use a concentration of 5 µg/ml.
WB 1/500 - 1/1000. Detects a band of approximately 80 kDa (predicted molecular weight: 89 kDa).

Target

  • Function

    ATP-dependent protease which is essential for axonal development.
  • Tissue specificity

    Ubiquitous. Highly expressed in the cerebellar Purkinje cells.
  • Involvement in disease

    Defects in AFG3L2 are the cause of spinocerebellar ataxia type 28 (SCA28) [MIM:610246]. It 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. SCA28 is an autosomal dominant cerebellar ataxia (ADCA) with a slow progressive course and no evidence of sensory involvement or cognitive impairment.
    Defects in AFG3L2 are the cause of spastic ataxia autosomal recessive type 5 (SPAX5) [MIM:614487]. A neurodegenerative disorder characterized by early onset spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy.
  • Sequence similarities

    In the N-terminal section; belongs to the AAA ATPase family.
    In the C-terminal section; belongs to the peptidase M41 family.
  • Cellular localization

    Mitochondrion membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • AFG3 (ATPase family gene 3, yeast) like 2 antibody
    • AFG3 ATPase family gene 3 like 2 (yeast) antibody
    • AFG3 ATPase family gene 3 like 2 antibody
    • AFG3 like protein 2 antibody
    • AFG3-like protein 2 antibody
    • AFG32_HUMAN antibody
    • AFG3L2 antibody
    • ATPase family gene 3 like 2 antibody
    • ATPase family gene 3 yeast antibody
    • EC 3.4.24.- antibody
    • FLJ25993 antibody
    • Paraplegin like protein antibody
    • Paraplegin-like protein antibody
    • SCA28 antibody
    • Spinocerebellar ataxia 28 antibody
    see all

Images

  • All lanes : Anti-AFG3L2 antibody (ab68023) at 1/500 dilution

    Lane 1 : AFG3L2 transfected 293T cell lysate
    Lane 2 : Non transfected 293T cell lysate

    Lysates/proteins at 25 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG (H&L)-HRP Conjugate secondary
    antibody at 1/2500 dilution

    Developed using the ECL technique.

    Predicted band size: 89 kDa
    Observed band size: 80 kDa
    why is the actual band size different from the predicted?

  • ICC/IF image of ab68023 stained HepG2 cells. The cells were 4% formaldehyde 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 (ab68023, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 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:

  • Boutoual R  et al. Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPAR?-UCP2-AMPK axis. Sci Rep 8:1163 (2018). Read more (PubMed: 29348686) »
  • Cesnekova J  et al. Loss of Mitochondrial AAA Proteases AFG3L2 and YME1L Impairs Mitochondrial Structure and Respiratory Chain Biogenesis. Int J Mol Sci 19:N/A (2018). Read more (PubMed: 30544562) »
See all 5 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

I have initiated the replacement order for one vial of ab17995. You should receive it shortly. Please let me know if you need anything further.

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Answer

Thanks for your email.

Yes, I will be able to send a vial of ab17995 to replace the original vial of ab68023. However I was unable to locate the order in our system. Do you any other order numbers or Abcam order reference numbers associated with it? What date was the order placed?

Thanks for the additional information!

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Answer

Thanks for submitting your completed questionnaire.

I am sorry this vial of ab68023 is giving you trouble. The protocol seems fine and the loading control staining worked well. So it seems there is an issue with the vial of antibody you received. If you have received this antibody within the past 6 months I am happy to offer a replacement, credit or refund.

In your reply, please include the Abcam order referenc number or PO number associated with this purchase so I can process your request.

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Answer

Thank you for your response and yoru comment.

This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab68023. For your information, the internal reference note for this credit is CN19981. You can use this credit in the near future for any of the products which are in the catalogue.

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 your response.

The datasheet suggests using AFG3L2 transfected 293T cell lysate as positive. Furthermore, the Western blot image describes that AFG3L2 transfected 293T cell lysate gave a signal at the expected size (around 80 kDa) whilst the non transfected 293T cell lysate did not give any signal.

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 your asking and letting us know the enquiry regarding ab68023 .

The antibody has been tested in AFG3L2 transfected 293T cell lysate as the datasheet indicates. We have no data/experience yet in using the antibody on other samples and it has not been tested on endogenously expressed samples.

I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question

LOT NUMBER GR67925-1 ORDER NUMBER 1002459 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE 293T cell PRIMARY ANTIBODY Concentration or dilution: 1:1000˜1:100 Diluent buffer : TBST Incubation time :o/n Incubation temperature: 4℃ Wash Buffer : PBST Number of washes : 10minX3times DETECTION METHOD ECL solution POSITIVE AND NEGATIVE CONTROLS USED - ANTIBODY STORAGE CONDITIONS -20C SAMPLE PREPARATION Lysis buffer RIPA buffer Protease inhibitors: inhibitor cocktail Phosphatase inhibitors NaF, sodium othovanadate, PMSF Boiling for ≥5 min? yes AMOUNT OF PROTEIN LOADED 30ug/lame ELECTROPHORESIS/GEL CONDITIONS Percentage of gel 6% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS Type of membrane:PVDF Protein transfer verified : semi-dry Blocking agent and concentration : 5% skim milk Blocking time : 1hr Blocking temperature : room temp. SECONDARY ANTIBODY Species: goat-anti mouse IgG-HRP Reacts against: mouse Concentration or dilution : 1:10000˜1:2500 Diluent buffer : TBST Incubation time : 2hr Incubation temperature: room temp. Fluorochrome or enzyme conjugate: HRP conjugate Wash Buffer : PBST Number of washes : 10minX3times HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? primary antibody concentration secondary antibody concentration Total protein and mitochondrial fraction concentration Blocking buffer : skim milk to 1% BSA ADDITIONAL NOTES The customer compare the data using the same secondary antibody and he said there's no problem with the seconday antibody.

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Answer

Thank you for your enquiry regarding ab68023 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

Could you please confirm if the customer is using transfected 293T cells? This antibody has been tested on AFG3L2 transfected 293T cell lysate. The non-transfected cells do not have any signal for AFG3L2 (please see the Western blot image on the datasheet).

I look forward to hearing from you soon.

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Application
Western blot
Sample
Human Cell lysate - whole cell (HEK-293, Hela)
Loading amount
20 µg
Specification
HEK-293, Hela
Gel Running Conditions
Reduced Denaturing (7%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Mr. Benny Garfinkel

Verified customer

Submitted Jul 08 2010

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