Anti-F-actin antibody [NH3] (ab205)

Reviews (8)Q&A (26)References (41)

Overview

  • Product name
    Anti-F-actin antibody [NH3]
    See all F-actin primary antibodies
  • Description
    Mouse monoclonal [NH3] to F-actin
  • Host species
    Mouse
  • Specificity
    Clone NH3 recognises human Filamentous actin (F-actin) and reportedly also recognizes Globular actin (G-actin). The antibody binds to the N-terminal region of actin, but not to the extreme N-terminal 40 amino acids. In tissue sections the antibody stains the cytoplasm of macrophages strongly, and gives granular, localised nuclear staining of all cell types. As this is a mouse IgM antibody, an anti-mouse IgM secondary antibody must be used to detect this primary.
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, IHC-R, WB, ELISA, IHC-Fr, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Human
  • Immunogen

    Human monocytes and U397 cell line.

  • Epitope
    This antibody is reported to recognise actin in the filamentous form with the epitope likely to be located between residues 120 and 226 of the molecule.
  • Positive control
    • gastrointestinal tissue

Properties

Applications

Our Abpromise guarantee covers the use of ab205 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
Flow Cyt 1/10.

ab91545 - Mouse monoclonal IgM, is suitable for use as an isotype control with this antibody.
ICC/IF 1/100.
IHC-R Use at an assay dependent concentration.
WB 1/100 - 1/500. Detects a band of approximately 43 kDa. As this is a mouse IgM antibody, an anti-mouse IgM secondary antibody must be used to detect this primary.
ELISA 1/10.
IHC-Fr Use at an assay dependent concentration.
IP Use at an assay dependent concentration. PubMed: 27336173

Target

Images

  • Western blot - Anti-F-actin antibody [NH3] (ab205)
    Western blot - Anti-F-actin antibody [NH3] (ab205)This image is courtesy of an anonymous Abreview
    Anti-F-actin antibody [NH3] (ab205) at 1/100 dilution + Human pancreatic cancer cell line, whole cell lysate at 20 µg

    Secondary
    HRP conjugated goat anti-mouse antibody at 1/2000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 43 kDa
    why is the actual band size different from the predicted?


    Exposure time: 1 minute

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-F-actin antibody [NH3] (ab205)
    Immunocytochemistry/ Immunofluorescence - Anti-F-actin antibody [NH3] (ab205)Image from Ibrahim, Ahmed S. et al. Oncotarget 7.8 (2016): 8532–8545. doi: 10.18632/oncotarget.7384, Fig 6D.

    Immunocytochemistry/ Immunofluorescence analysis of ARPE cells labelling F-actin with ab205. Cells were fixed in 4% paraformaldehyde for 10 min, washed with PBS, and blocked for one hour. Cells were then incubated for 3h at 37°C with Anti-F-actin antibody [NH3] (ab205) at 1/200 dilution. Thereafter, cells were washed 3 times with PBS containing 0.3% Triton-X, incubated with secondary antibody and coverslipped with Fluoroshield containing DAPI as a counter stain. Immunofluorescent signals were detected by confocal microscopy.

  • Immunohistochemistry (Resin sections) - Anti-F-actin antibody [NH3] (ab205)
    Immunohistochemistry (Resin sections) - Anti-F-actin antibody [NH3] (ab205)This image is courtesy of an anonymous abreview.

    Immunohistochemistry (Resin sections) analysis of mouse bone tissue cells labeling F-actin with ab205 at 1/200 dilution. Mouse tibia bone samples were fixed in neutral buffered formalin for 72 hours at room temperature, embedded undecalcified in plastic and sectioned at 5 microns.  A polyclonal goat anti-mouse Alexa Fluor® 488 secondary antibody was used at 1/700 dilution. The image shows a 1 micron thick z-projection of the chondrocytes in the proximal tibia growth plate. 

    See Abreview

  • Western blot - Anti-F-actin antibody [NH3] (ab205)
    Western blot - Anti-F-actin antibody [NH3] (ab205)This image is courtesy of an anonymous abreview.
    Anti-F-actin antibody [NH3] (ab205) at 1/500 dilution + CNS whole tissue lysate at 30 µg

    Secondary
    Goat anti-mouse HRP conjugate at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 43 kDa why is the actual band size different from the predicted?


    Exposure time: 5 minutes


    Blocking: 5% milk for 1 hour at 25°C. 

    Incubation with primary antibody for 15 hours at 4°C in 5%milk+ PBS+0.1% Tween 20.

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-F-actin antibody [NH3] (ab205)
    Immunocytochemistry/ Immunofluorescence - Anti-F-actin antibody [NH3] (ab205)This image is courtesy of an anonymous abreview.

    Immunocytochemistry/ Immunofluorescence analysis of mouse cortical neurons labeling F-actin with ab205 at 1/100 dilution. The cells were fixed with Ethanol and permeabilized with 0.2% Triton X-100. A polyclonal goat anti-mouse Cy3 conjugated secondary antibody was used at 1/200 dilution. 

    See Abreview

  • Immunohistochemistry (Frozen sections) - Anti-F-actin antibody [NH3] (ab205)
    Immunohistochemistry (Frozen sections) - Anti-F-actin antibody [NH3] (ab205)Image courtesy of an Abreview submitted by Jamie Richards.
    ab205 staining F-actin in rat brain tissue by Immunohistochemistry (frozen sections). Tissue was fixed with paraformaldehyde and then blocked with 2% BSA for 1 hour followed by incubation with the primary antibody at a 1/25 dilution for 24 hours. A biotin-conjugated goat anti-mouse IgM was used as secondary antibody at a 1/400 dilution. No permeabilisation step was used.

References

This product has been referenced in:
  • Shi C  et al. Yap promotes hepatocellular carcinoma metastasis and mobilization via governing cofilin/F-actin/lamellipodium axis by regulation of JNK/Bnip3/SERCA/CaMKII pathways. Redox Biol 14:59-71 (2018). Read more (PubMed: 28869833) »
  • Yan H  et al. Yap regulates gastric cancer survival and migration via SIRT1/Mfn2/mitophagy. Oncol Rep 39:1671-1681 (2018). Read more (PubMed: 29436693) »
See all 41 Publications for this product

Publishing research using ab205? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 34 Abreviews or Q&A

Immunohistochemistry (Frozen sections) abreview for Anti-F-actin antibody [NH3]

 Good
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue (Pancreas PanIN)
Permeabilization
No
Specification
Pancreas PanIN
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Fixative
Methanol
Read More

Abcam user community

Verified customer

Submitted Jun 27 2018

Immunocytochemistry/ Immunofluorescence abreview for Anti-F-actin antibody [NH3]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Mouse Derived Breast cancer Cell Line)
Permeabilization
Yes - 0.5 % Triton
Specification
Mouse Derived Breast cancer Cell Line
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 26°C
Fixative
Methanol
Read More

Abcam user community

Verified customer

Submitted Aug 17 2017

Western blot abreview for Anti-F-actin antibody [NH3]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mouse Derived Breast Cancer Primary Cell Line)
Gel Running Conditions
Reduced Denaturing (10)
Loading amount
40 µg
Specification
Mouse Derived Breast Cancer Primary Cell Line
Blocking step
Licor Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
Read More

Abcam user community

Verified customer

Submitted Aug 14 2017

Immunocytochemistry/ Immunofluorescence abreview for Anti-F-actin antibody [NH3]

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Cortical Neurons)
Permeabilization
Yes - 0.2% Triton X-100
Specification
Cortical Neurons
Blocking step
casein solution + Tween 20 as blocking agent for 20 minute(s) · Concentration: 0.2% · Temperature: 25°C
Fixative
Ethanol
Read More

Abcam user community

Verified customer

Submitted Aug 04 2016

Question

Je suis à la recherche d’un anticorps spécifique de l’actine F et un autre spécifique de l’actine monomérique ou actin-G.

J’aimerais un anticorps déjà testé en Immunofluorescence et pas de toxine type phalloidin.

Read More
Answer

Je peux vous proposer ab205, Anti-F-actin antibody [NH3] qui est un monoclonal de souris garanti de fonctionner en immunofluorescence. Cependant, nous n’avons pas testé le taux de toxine dans ce produit et donc nous ne pouvons pas garantir qu’il n’y a pas de toxine type phalloidin présent.


Malheureusement, nous n’avons pas d’anticorps anti-G-actine. Tous nos autres anticorps reconnaissent les deux formes F et G.

Read More

Western blot abreview for Anti-F-actin antibody [NH3]

 Excellent
Abreviews
abreview image
Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Tissue lysate - whole (CNS)
Specification
CNS
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Abcam user community

Verified customer

Submitted Aug 01 2014

Immunohistochemistry (Resin sections) abreview for Anti-F-actin antibody [NH3]

 Excellent
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
abreview image
Application
Immunohistochemistry (Resin sections)
Sample
Mouse Tissue sections (Bone Tissue Cells)
Specification
Bone Tissue Cells
Read More

Abcam user community

Verified customer

Submitted Jul 10 2013

Question

I got the reply from the customer in regards to ab205:
"I have tried, according to your suggestion, to do the IHC experiment again with a different secondary antibody "Alexa Fluor® 488 Goat Anti-Mouse IgG, IgM (H+L)" that recognize both IgG and IgM. This didn't recognize the F-actin antibody and resulted in no positive-specific staining.
At this point I think I have tried almost every step in the attempts to get a positive signal."

Looking forward to your reply.
Thanks in advance for your assistance.

Best Regards,

Read More
Answer

Thank you for your message and for providing this further information.
I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or refund in compensation.
If you decide to get a free of charge replacement we would recommend it would be beneficial to include a positive control, such as gastrointestinal tissue or brain tissue in the following experiments.
I look forward hearing from you with details of how you would like to proceed.

Read More

Question

Please read below customer's response to your comments about ab205 [CCE4313628] and also about ab128190 [CCE4318992]:
"Thank you for your answer and suggestions. i have addressed all of your suggestions as follows
1.The secondary antibody that we are using recognize all of the isotope of the mouse heavy chains. i attached to the original IHC compliment form the positive control which was a mouse anti mouse antibody against PCNA ( https://www.abcam.com/PCNA-antibody-PC10-Proliferation-Marker-ab29.pdf ). the secondary antibody recognized it so surely i can say that it recognize the isotope.
2.Although it is not a part of a standard staining protocol in frozen sections, i performed an heat-induced epitope retrieval according to your suggestion as follows: the sections were heated (microwave) in 0.01M sodium citrate for 15 minutes total. Full power until reaching boiling point and then 20% form the maximum power for the remaining time.
the result was again no positive staining
looking forward to receiving your reply"
Please advise.

Thanks in advance for your assistance and reply.

Read More
Answer

Thank you for taking the time to provide additional information and to include our suggestions in your experiments. I appreciate the time that was spent doing additional experiments. I am sorry to hear that the results obtained are still not satisfactory.
Reviewing the details you have kindly provided gave us more insight in this case.
I can confirm that the Isotype of the Anti-F-actin antibody [NH3] (ab205) is an IgM. The secondary antibody listed on the form provided as far as I can see detects all isotypes of IgG. For that reason it will work with the Anti-PCNA antibody [PC10] - Proliferation Marker (ab29) that was mentioned. However I would regrettably not expect it to detect the ab205 - IgM. Could you confirm that this was the secondary antibody that was used? I would suggest repeating the experiment with a secondary antibody that detects IgM. If still no satisfactory results are obtained please do not hesitate to contact us again and I am pleased to provide a replacement.
While more information were provided in the previous email the Anti-Cytokeratin 5 antibody [2C2] (ab128190) was mentioned. Could you please confirm if there are problems with this antibody? If so we would be pleased to investigate this case further for you.
Could you please provide information about the experiment with the Anti-Cytokeratin 5 antibody [2C2] (ab128190) and the results by filling in a new questionnaire? That will help us to investigate the case for our quality control. We appreciate the time spent on this and hope that we can be of help.
I look forward hearing from you with the requested details. I hope we can resolve the case.

Read More

Question

Dear Tech Support Team,
Kindly advise in regards to the following information. The images are attached.

Order Details:
Antibody code: ab205 Anti F-Actin.
Batch number: GR3830-12

General Information:

Antibody storage conditions (temperature/reconstitution etc)
The Antibody was aliquoted and stored at -20C immediately after arrival.

Description of the problem (high background, low signal, non-specific staining etc.)
lack of stain, there is no positive stain at all.

Sample (Species/Tissue/Cell Type/Cell Line etc.)
the samples are 7 µm thick frozen sections from mouse testis embedded in OCT.

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
4% paraformaldehyde for 2 days followed by OCT embedding.

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
No antigen retrieval is necessary in frozen sections

Permeabilization step
20 min incubation with 4% paraformaldehyde for each section on slides.

Blocking conditions (Buffer/time period, Blocking agent etc.)
1 h in buffer (10% FCS, 0.5% BSA 1% triton. 88.5% PBS) at room temperature.

Primary Antibody (Diluent/Dilution/Incubation time, Wash step)
1:100 and 1:50 dilution in the blocking solution. over night incubation 4C in a closed humified box.

Washes were performed in PBST (PBS buffer+TWEEN) 0.2%, for 5 minutes twice.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
the secondary antibody was "Alexa Flour 488 Goat Anti-Mouse IgG (H+L), Molecular Probes A11001". I used it at 1:100 dilution, in the blocking solution.

Detection method
Fluorescence.

Positive and negative controls used (please specify)
for negative control, only secondary (no primary) antibody was used.
For positive control, another stain was performed using abcam's anti PCNA antibody (ab-29) and the same reagents, incubation times and secondary antibody.

Optimization attempts (problem solving):
Different dilutions for the primary antibody.
Longer incubation times for the primary antibody and for the secondary antibody as well.

How many times have you tried the IHC?
4 times.

Have you run a "No Primary" control?
yes

Do you obtain the same results every time?
Yes, no staining signal every time.

What steps have you altered?
Different dilutions for the primary antibody.
Shorter wash times.
Longer incubation times for the primary antibody and for the secondary antibody as well.

Thanksin advance fro your assistance and reply.
Best Regards,

Read More
Answer

Thank you for contacting us.
I am sorry to hear you have been experiencing problems with one of our Anti-F-actin antibody [NH3] (ab205). The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints. I appreciate the time taken to submit further information to us, and after reading the answers provided I would like to ask some additional questions.
1. Could you please confirm the isotope recognition of the secondary antibody by double checking with the customer? I ask to make sure that there are no incompatibility issues with our primary Anti-F-actin antibody.
2. The protocol shows two fixation steps. It could be that the double fixation masks the immunogen for the primary antibody. Could you confirm if washing steps before the primary antibody incubation include some kind of permeabilization? If not we would suggest to try a mild antigen retrieval (heat-induced epitope retrieval, or HIER) using the microwave. A protocol can be found under this provided link:
https://www.abcam.com/ps/pdf/protocols/ihc_p.pdf
I hope some of the suggestions may help to solve the issue and to improve the results. As the antibody was purchased in the past 120 days 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.
I look forward to receiving your reply.

Read More

1-10 of 34 Abreviews or Q&A

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