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Validated using a knockout cell line

Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)

Reviews (3)Q&A (6)References (21)
Western blot - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
  • Immunocytochemistry/ Immunofluorescence - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
  • Western blot - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
  • Flow Cytometry - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)

Key features and details

  • Mouse monoclonal [5E2D7] to ATPase Inhibitory Factor 1/IF1
  • Suitable for: ICC/IF, Flow Cyt, WB
  • Knockout validated
  • Reacts with: Mouse, Rat, Cow, Human
  • Isotype: IgG1

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647

Overview

  • Product name

    Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7]
    See all ATPase Inhibitory Factor 1/IF1 primary antibodies

  • Description

    Mouse monoclonal [5E2D7] to ATPase Inhibitory Factor 1/IF1

  • Host species

    Mouse

  • Tested applications

    Suitable for: ICC/IF, Flow Cyt, WBmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human

  • Immunogen

    Recombinant full length protein. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • Isolated mitochondria from Human heart, Bovine heart, Rat heart and Mouse heart IF/ICC: HepG2 cell line.

  • General notes

    This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

    Product was previously marketed under the MitoSciences sub-brand.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab110277 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 (1)
Use a concentration of 10 µg/ml.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB (1)
Use a concentration of 1 µg/ml. Detects a band of approximately 10,18 kDa (predicted molecular weight: 12 kDa).
Notes
ICC/IF
Use a concentration of 10 µg/ml.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB
Use a concentration of 1 µg/ml. Detects a band of approximately 10,18 kDa (predicted molecular weight: 12 kDa).

Target

  • Function

    Endogenous F(1)F(o)-ATPase inhibitor limiting ATP depletion when the mitochondrial membrane potential falls below a threshold and the F(1)F(o)-ATP synthase starts hydrolyzing ATP to pump protons out of the mitochondrial matrix.

  • Sequence similarities

    Belongs to the ATPase inhibitor family.

  • Post-translational
    modifications

    Exhibits variability in chain length, mitochondria have distinct pools of protein cleaved after the 24th, 25th, and 26th amino acid.

  • Cellular localization

    Mitochondrion.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • ATIF1_HUMAN antibody
    • ATP synthase inhibitor protein antibody
    • ATPase inhibitor antibody
    • ATPase inhibitor mitochondrial antibody
    • ATPase inhibitor protein antibody
    • ATPase inhibitory factor 1 antibody
    • ATPI antibody
    • ATPIF 1 antibody
    • Atpif1 antibody
    • ATPIP antibody
    • IF(1) antibody
    • IF1 antibody
    • Inhibitor of F(1)F(o)-ATPase antibody
    • IP antibody
    • MGC1167 antibody
    • MGC8898 antibody
    • mitochondrial antibody
    see all

Images

  • Western blot - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    Western blot - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    All lanes : Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277) at 1 µg/ml

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : ATPase Inhibitory Factor 1 / IF1 knockout HAP1 whole cell lysate
    Lane 3 : HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 12 kDa



    Lanes 1 - 3: Merged signal (red and green). Green - ab110277 observed at 12 kDa. Red - loading control, ab181602, observed at 37 kDa.

    ab110277 was shown to recognize ATPase Inhibitory Factor 1 / IF1 in wild-type HAP1 cells as signal was lost at the expected MW in ATPase Inhibitory Factor 1 / IF1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and ATPase Inhibitory Factor 1 / IF1 knockout samples were subjected to SDS-PAGE. Ab110277 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    Immunocytochemistry/ Immunofluorescence - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)

    ICC/IF image of ab110277 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 (ab110277, 10µg/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

  • Western blot - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    Western blot - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    All lanes : Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277) at 1 µg/ml

    Lane 1 : Human heart mitochondria at 10 µg
    Lane 2 : Bovine heart mitochondria at 4 µg
    Lane 3 : Rat heart mitochondria at 10 µg
    Lane 4 : Mouse heart mitochondria at 10 µg

    Predicted band size: 12 kDa

  • Flow Cytometry - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    Flow Cytometry - Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277)
    Overlay histogram showing HepG2 cells stained with ab110277 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab110277, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab91353) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References (21)

Publishing research using ab110277? Please let us know so that we can cite the reference in this datasheet.

ab110277 has been referenced in 21 publications.

  • Kahancová A  et al. Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells. Sci Rep 10:1551 (2020). PubMed: 32005857
  • Anand R  et al. MIC26 and MIC27 cooperate to regulate cardiolipin levels and the landscape of OXPHOS complexes. Life Sci Alliance 3:N/A (2020). PubMed: 32788226
  • Li J  et al. Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS. EMBO Rep 21:e48781 (2020). PubMed: 31916354
  • Golic I  et al. Insulin Modulates the Bioenergetic and Thermogenic Capacity of Rat Brown Adipocytes In Vivo by Modulating Mitochondrial Mosaicism. Int J Mol Sci 21:N/A (2020). PubMed: 33287103
  • Kovalcíková J  et al. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme. FASEB J 33:14103-14117 (2019). PubMed: 31652072
View all Publications for this product

Customer reviews and Q&As

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1-9 of 9 Abreviews or Q&A

Western blot abreview for Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Rat Cell lysate - whole cell (rat primary cardiac fibroblasts)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
30 µg
Specification
rat primary cardiac fibroblasts
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Dec 28 2021

Immunocytochemistry/ Immunofluorescence abreview for Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7]

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (rat primary cardiac fibroblasts)
Permeabilization
Yes - 1% Triton 4 min
Specification
rat primary cardiac fibroblasts
Fixative
Paraformaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Dec 07 2021

Immunoprecipitation abreview for Anti-ATPase Inhibitory Factor 1 / IF1 antibody [5E2D7]

 Excellent
Abreviews
abreview image
Application
Immunoprecipitation
Sample
Mouse Cell lysate - whole cell (EL4)
Total protein in input
2e+006 cells
Immuno-precipitation step
Protein G
Specification
EL4
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Dr. Michael Berger

Verified customer

Submitted Apr 27 2018

Question

It seems quite strange to me that the fact that this antibody also binds to a subunity of Cyt c oxidase is not reported in the datasheet. Could you please tell me what the MW of this subunit is?

Read More
Answer

Please find attached a document showing a very detailed analysis of IF1 antibody. This work was done many years ago by a scientist in our laboratory. Although the datasheet does not say the antibody is IP+, the researchers found it to be IP+ and show that most of the bound protein to the IF1 antibody was released from the complex V under a 55 -25% sucrose gradient. The 18 kDa band however was found to be tightly attached to COX and later found to be in fact COXIV (see section 3, 6 and 7).

Read More

Question

I would like to visualize IF1 inhibitory subunit of ATPase in mitochondria of different cell types by confocal microscopy. The datasheet shows two bands (of about 10 KDa and 20 KDa) in human and bovine mitochondria and only one band (of about 20 KDa) in mouse and rat mitchondria. What do you think of this higher band, considering that the molecular weight of IF1 is about 12 KDa and why there is no lower band in rat and mouse mitochondria?

Read More
Answer

-In regards to WB and IP applications, this antibody is very useful to monitor IF1 as you can differentiate between the 10 and 18kDa band. Ratios of the two bands vary from preparation to preparation of homogenate/mitochondria even in the same species. There is also a lot of variation within species. It has been suggested in the literature that IF1 varies in response to ischemia and this may account for the variable ratios of the bands. Therefore we believe that variability may depend on how the tissue is handled prior to protein extraction or isolation of mitochondria. In regards to ICC type of applications, it will not be possible to distinguish between the two proteins.


-It is conceivable that tissue preparation at high pH would shift the bandage towards the 18kDa (COXIV) and that lowering the pH to 6.5 may help the shift towards the 10kDa (IF1), although this is speculative.

Read More

Question

Product code: 110277
Inquiry: Technical Inquiry about product MS506 (ab110277) Mitosciences Lot#B0372 Which kind of immunogen have you used to rise the primary ab MS506? Recombinant full lenght protein (UniProtKB ID: Q6IAQ7) the protein itself without mitoch transition peptide, or just a sub-sequence (which one)? Under our experimental conditions more than the two already reported bands do appear (and are very strong) with an MW higher than 25kDa (range 25 to 65 kDa) IF1 has been reported indeed to appear also as a dimer under SDS-PAGE standard conditions (MW approx 24kDa PLoS September 2011 | Volume 6 | Issue 9 | e23949 [primary Ab rabbit polyclonal home made]) Seemingly the only IF1 specific band in our samples is that of approx 11kDa, the upper does not match with a standard IF1 peptide used as a reference Thank you for your kind attention Experimental conditions used WB using primary Ab cat number MS506 Samples: mitochondrial fraction 10 to 40ug / lane cells type: human hepatocarcinoma (lines: HepG2 and JHH-6) band resolved with: standard SDS-PAGE (%T:17% acril to bis-acril ratio: 37,5:1) under reducing conditions (beta-mercapto) gel run at 20mA constant / each gel Blotting: (full wet 1h30min 250V) blocking solution: BSA 5% in PBS-T (Tween-20 is 0,1%) 1h 4°C primary Ab: MS506 (1:1500) in blocking solution ON 4°C secondary Ab: HRP-conjugated (1:5000) in blocking solution 1h RT linear signal tested for each sample from 10 to 40 ug of mitoch proteins and for each exposure time (reagents: Dura lum+Dura perox images acquired with a Biorad ChemidocXRS apparatus)

Read More
Answer

Thank you for contacting us.

The immunogen of this antibody does contain the transit peptide.

This antibody actually targets on an epitope that exists both in ATP- IF and COX IV subunits, which means the double bands showed on the WB are COX IV (upper) and IF (lower). The strength of the two bands is variant dramatically from species to species.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Read More

Question

I have received the response from this customer:

1) What did you load into the different lanes?
Allare the mitochondrial extracts of mouse liver. Left three lanes: wild type control,rightthreelanes: miRNA 122knock-outmice.

2) Have you tried any other sample as a positive control, e.g. isolated mitochondria from human, bovine, rat or mouse heart?
He has not tried other sample, and I have reminded him may to try.

Besides, he would like to confirm which lane is the target band? Around 20 or 17kDa?

Read More
Answer

Thank you for your reply, and the additional information.

According to your description of the lanes in your WB, are you expected to see no bands in the 3 right lanes- due to the miRNA knout out? Please let me know.

I would also recommend trying a positive control, as I had mentioned in my recent email.

The target band would be theone with the smallest MW, 17 kDa, I suppose. I was not able to read that MW number that's why I didnot specifically state the molecular weight. I apologize.

But again, if the antibody was purchased within the last 6 months, we can offer a replacement, credit or refund if your arenot satisfied with the antibody. In that case, please let me know your order or PO number.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Question

After contacting this customer, he provides some information:

1) A WB image would be very helpful : attached please find
2) How much sample was loaded onto the gel? 30ug protein
3) What antibody dilution and incubation time was used? ab110277 :1/1000, 18hr in 4°C; 2nd Ab 1/5000, RT 1hr
4) Have you tried a no primary control in order to check if the secondary antibody might be contributing to the additional bands? No, but the same secondary Ab works well with other primary Ab.
5) What was sued for blocking? 5% non-fat-milk in TBST



Would you please help him check the data?

Read More
Answer

Thank you for your reply and the addtional information including the image.

The strong band at 26 kDa is probably endogenous IgG light chain -since you are using a tissue sample this could be likely. A band like this should disappear when using a cell line

We have seen aband for the mouse and rat protein at 18 kDa with mitochondrial heart lysate. Thus, the lower band you see could be the correct band.

I was also wondering:
1) What did you load into the different lanes?
2) Have you tried any other sample as a positive control, e.g. isolated mitochondria from human, bovine, rator mouse heart?

If the antibody was purchased within the last 6 months, we can offer a replacement, credit or refund if the customer is not satisfied with the antibody. In that case, please let me know your order or PO number.

I look forward to hear back and assist you further.

Read More

Question

Our customer has purchased ab110277: Anti-ATPase Inhibitory Factor 1 antibody [5E2D7] and conducted WB with mitochondria extraction from mouse liver sample. There are three bands detected around 26(the strongest), 20, and 16 kDa. The results confused him and the data is needed urgently. Therefore, he sincerely would like to know which the target signal is. I am glad to provide the results to analyse.

Would you please help him resolve this problems?

Read More
Answer

Thank you for contacting us.

I am sorry to hear that the customer sees multiple bands.
I will check with the lab regarding the bands they saw for mouse samples and let you know.

In the meantime, could you please send me the following information:

1) A WB image would be very helpful.
2) How much sample was loaded onto the gel?
3) What antibody dilution and incubation time was used?
4) Have you tried a no primary control in order to check if the secondary antibody might be contributing to the additional bands?
5) What was sued for blocking?

I look forward to hear back from you, and will be in touch regarding the lab'sresponse as well.

Read More

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