Overview

  • Product name
    Anti-TOMM20 antibody - Mitochondrial Marker
    See all TOMM20 primary antibodies
  • Description
    Rabbit polyclonal to TOMM20 - Mitochondrial Marker
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat, Orangutan
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human TOMM20.

    Read Abcam's proprietary immunogen policy (Peptide available as ab88207.)

  • Positive control
    • This antibody gave a positive signal in HepG2 whole cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab78547 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 1 µg/ml.
IP Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 16 kDa (predicted molecular weight: 16 kDa).
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function
    Central component of the receptor complex responsible for the recognition and translocation of cytosolically synthesized mitochondrial preproteins. Together with TOM22 functions as the transit peptide receptor at the surface of the mitochondrion outer membrane and facilitates the movement of preproteins into the TOM40 translocation pore.
  • Sequence similarities
    Belongs to the Tom20 family.
  • Cellular localization
    Mitochondrion outer membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • KIAA0016 antibody
    • MAS20 antibody
    • MGC117367 antibody
    • Mitochondrial 20 kDa outer membrane protein antibody
    • Mitochondrial import receptor subunit TOM20 homolog antibody
    • MOM19 antibody
    • Outer mitochondrial membrane receptor Tom20 antibody
    • TOM20 antibody
    • TOM20_HUMAN antibody
    • TOMM20 antibody
    • Translocase of outer mitochondrial membrane 20 homolog (yeast) antibody
    • Translocase of outer mitochondrial membrane 20 homolog type II antibody
    see all

Images

  • ICC/IF image of ab78547 stained HepG2 cells. The cells were 4% PFA 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 (ab78547, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, Hek293, MCF-7 cells at 1µg/ml, and in 100% Methanol fixed (5 min) HepG2, HeLa, Hek293, MCF-7 cells at 1µg/ml.
  • Anti-TOMM20 antibody - Mitochondrial Marker (ab78547) at 1 µg/ml + HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 16 kDa
    Observed band size: 16 kDa
    Additional bands at: 70 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 30 seconds
  • IHC image of TOMM20 staining in Human Liver carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab78547, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • TOMM20 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to TOMM20 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab78547.
    Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.
    Band: 16kDa; TOMM20.

References

This product has been referenced in:
  • Liu H  et al. Mitophagy protects SH-SY5Y neuroblastoma cells against the TNFa-induced inflammatory injury: Involvement of microRNA-145 and Bnip3. Biomed Pharmacother 109:957-968 (2019). Read more (PubMed: 30551550) »
  • Kumar S  et al. Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins. J Cell Biol 217:997-1013 (2018). Read more (PubMed: 29420192) »
See all 24 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (human fibroblast mitochondria)
Gel Running Conditions
Reduced Denaturing
Loading amount
100 µg
Specification
human fibroblast mitochondria
Blocking step
BSA as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jul 27 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (mouse embryonic fibroblast)
Specification
mouse embryonic fibroblast
Fixative
Formaldehyde
Permeabilization
Yes - 0.5% Triton X
Blocking step
BSA and image-iTFX (invitrogen) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 15 2013

Question

Thank you for your recent reply. I have attahed the blot as a pdf, I hope this is readable. I have also filled in the details below:


Order Details
Antibody code: ab 78547

Problem
Choose: weak signal

Lot number: 874838

Purchase order number
or preferably Abcam order number: Sent as a replacement to ab13252



General Information
Antibody storage conditions (temperature/reconstitution etc)

Aliquots at 4C and -20C

Description of the problem (high background, wrong band size, more bands, no band etc.)
Very weak signal, more bands

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)

Whole cell lysate

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

RIPA buffer


Amount of protein loaded
25-50ug

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
17.5% SDS-PAGE gel

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)

Transfer 1hr, blocking 1hr milk

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Used at recommended dilution 1:600 overnight. Washed 3x 5min PBS+0.1% Tween

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Anti-rabbit HRP dilution 1:15,000 1hr incubation. Washed 3x10min PBS+0.1% Tween

Detection method (ECL, ECLPlus etc.)
ECL

Positive and negative controls used (please specify)
Positive control: large vol protein from whole cell lysates

Negative control: not relevant


Optimization attempts (problem solving)
How many times have you tried the Western?
At least 5x


Have you run a "No Primary" control?
No

Do you obtain the same results every time?
Yes

e.g. are the background bands always in the same place?


What steps have you altered?
Increased SDS-PAGE gel %, increased incubation with primary duration

Additional Notes:

Read More
Answer

Thank you for your response.

My colleague is out of office this week and she has asked me to look after her customers.

There seems to be a faint band at 15 kDa and another stronger one at 60 kDa in the Western blot image you have sent to us.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

TOMM20 is localised to mitochondrion outer membrane. In order to be able to increase the intensity of the lower band, it may be worth making mitochondrial preparation and increasing the final working concentration of this antibody (i.e. 1/500 or 1/300).

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Question

Thank you for your recent reply. I have attahed the blot as a pdf, I hope this is readable. I have also filled in the details below:

Order Details
Antibody code: ab 78547

Problem
Choose: weak signal



Lot number: 874838

Purchase order number
or preferably Abcam order number: Sent as a replacement to ab13252



General Information
Antibody storage conditions (temperature/reconstitution etc)

Aliquots at 4C and -20C

Description of the problem (high background, wrong band size, more bands, no band etc.)
Very weak signal, more bands

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)

Whole cell lysate

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

RIPA buffer


Amount of protein loaded
25-50ug

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
17.5% SDS-PAGE gel

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)

Transfer 1hr, blocking 1hr milk

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Used at recommended dilution 1:600 overnight. Washed 3x 5min PBS+0.1% Tween

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Anti-rabbit HRP dilution 1:15,000 1hr incubation. Washed 3x10min PBS+0.1% Tween

Detection method (ECL, ECLPlus etc.)
ECL

Positive and negative controls used (please specify)
Positive control: large vol protein from whole cell lysates

Negative control: not relevant


Optimization attempts (problem solving)
How many times have you tried the Western?
At least 5x


Have you run a "No Primary" control?
No

Do you obtain the same results every time?
Yes

e.g. are the background bands always in the same place?


What steps have you altered?
Increased SDS-PAGE gel %, increased incubation with primary duration

Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Read More
Answer

Thank you for your response.

My colleague Kate is out of office this week and she has asked me to look after her colleagues.

I understand that this vial was a free of charge replacement. Could you please summarize if the problem with ab78547 is similar to the original antibody (ab13252) CCE3506002?

This product has been tested for WB application on HepG2 and 1 µg/ml gave a distinctive band. Since the sample types (V2 and 293T cells) are different I would suggest testing higher concentration.

TOMM20 is localised to mitochondrion outer membrane. Have you tried using enriching TOMM20 and separating mitochondrial membranes rather than whole cell lysates?

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this second antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for human and WB. In the event that a product is not functioning in the tested applications and species 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.

As this is a different antibody, I would like to investigate thisindividually, and also obtain more information for our quality monitoring records as we did before. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I am sorry to confirm that regrettably theattachment sent is not readable. Iwould appreciate if youare able to send this image again as a JPEG file asitwould help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Formaldehyde
Permeabilization
Yes - 0.1% Triton X-100/PBS
Blocking step
0.5% BSA, 0.2% fish skin gelatin/PBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 28 2010

Abreviews
Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Xenopus laevis Cell lysate - whole cell (meiotic egg extract)
Loading amount
25 µg
Specification
meiotic egg extract
Gel Running Conditions
Reduced Denaturing (15%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 21 2010

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

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