Overview

  • Product name

  • Description

    Rabbit polyclonal to Cofilin
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-FoFr, ICC/IF, WB, IHC-Pmore details
  • Species reactivity

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

    Synthetic peptide corresponding to Human Cofilin aa 150 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab42823)

  • Positive control

    • WB: Recombinant Human Cofilin protein (ab62958), HeLa, Jurkat, A431, HEK293, MCF7, SHSY-5Y, PC12 and NIH 3T3 whole cell lysates. ICC/IF: HeLa cells. IHC-P: Human breast carcinoma tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab42824 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
IHC-FoFr 1/2000 - 1/3000.
ICC/IF Use a concentration of 1 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 20 kDa (predicted molecular weight: 19 kDa).
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function

    Controls reversibly actin polymerization and depolymerization in a pH-sensitive manner. It has the ability to bind G- and F-actin in a 1:1 ratio of cofilin to actin. It is the major component of intranuclear and cytoplasmic actin rods.
  • Tissue specificity

    Widely distributed in various tissues.
  • Sequence similarities

    Belongs to the actin-binding proteins ADF family.
    Contains 1 ADF-H domain.
  • Post-translational
    modifications

    Phosphorylated on Ser-3 in resting cells.
  • Cellular localization

    Nucleus matrix. Cytoplasm > cytoskeleton. Almost completely in nucleus in cells exposed to heat shock or 10% dimethyl sulfoxide.
  • Information by UniProt
  • Database links

  • Alternative names

    • 18 kDa phosphoprotein antibody
    • CFL 1 antibody
    • CFL antibody
    • CFL1 antibody
    • COF1_HUMAN antibody
    • Cofilin 1 antibody
    • Cofilin 1 non muscle antibody
    • Cofilin antibody
    • Cofilin non muscle isoform antibody
    • Cofilin-1 antibody
    • epididymis secretory protein Li 15 antibody
    • HEL-S-15 antibody
    • non-muscle isoform antibody
    • p18 antibody
    see all

Images

  • All lanes : Anti-Cofilin antibody (ab42824) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 4 : HEK293 Human embryonic kidney cell line Whole Cell Lysate
    Lane 5 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 6 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 19 kDa
    Observed band size: 20 kDa
    why is the actual band size different from the predicted?

  • ICC/IF image of ab42824 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab42824, 1µg/ml) for 1h at room temperature. 1% BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

  • IHC image of Cofilin staining in human breast carcinoma FFPE section, performed on a 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 ab42824, 1µg/ml, for 8 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.
  • Anti-Cofilin antibody (ab42824) at 1 µg/ml + Recombinant Human Cofilin protein (ab62958) at 0.01 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Predicted band size: 19 kDa

  • All lanes : Anti-Cofilin antibody (ab42824) at 1 µg/ml

    Lane 1 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
    Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 19 kDa
    Observed band size: 20 kDa why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab42824 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

References

This product has been referenced in:

  • Zhao F  et al. Exendin-4 promotes actin cytoskeleton rearrangement and protects cells from Nogo-A-?20 mediated spreading inhibition and growth cone collapse by down-regulating RhoA expression and activation via the PI3K pathway. Biomed Pharmacother 109:135-143 (2019). Read more (PubMed: 30396070) »
  • Yu HX  et al. MicroRNA-384 inhibits the progression of esophageal squamous cell carcinoma through blockade of the LIMK1/cofilin signaling pathway by binding to LIMK1. Biomed Pharmacother 109:751-761 (2019). Read more (PubMed: 30551528) »
See all 37 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (neurons)
Permeabilization
No
Specification
neurons
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 37°C
Fixative
Methanol

Abcam user community

Verified customer

Submitted Sep 18 2019

Application
Western blot
Sample
Mouse Tissue lysate - whole (muscle lysate)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
30 µg
Specification
muscle lysate
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jun 25 2019

Application
Western blot
Sample
Human Cell lysate - whole cell (Vascular smooth muscle cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
Vascular smooth muscle cells
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 25 2018

Application
Western blot
Sample
Pig Cell lysate - whole cell (isolated hepatocyte cells)
Gel Running Conditions
Reduced Denaturing (4-12% gradient gel)
Loading amount
10 µg
Specification
isolated hepatocyte cells
Blocking step
BSA as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 3% · Temperature: 20°C

Abcam user community

Verified customer

Submitted May 08 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Pig Tissue sections (endometrium)
Antigen retrieval step
Heat mediated
Permeabilization
No
Specification
endometrium
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 12 2016

Application
Western blot
Sample
Pig Tissue lysate - whole (endometrial tissue lysate)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
10 µg
Specification
endometrial tissue lysate
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Feb 11 2016

Question

Hi,
I am facing problem with one of your product Ab54532, all detail
is given below,
1) Abcam product code: ab54532
2) Abcam order reference number or product batch number:

3) Description of the problem:
We used this Product for Immunoprecipitation, to capture the
antigen in the cell culture supernatant then analyzed by western blot
by using ab42824 but there was no different between control(only
treated with Magnetic beads) and samples (treated magnetic beads and
ab54532). The Product ab42824 is working very well for normal western
blot. Then we tried Ab54532 for normal western blot by loading 50ug of
protein extract, found that not working from even from 2000-250
dilution. But product Ab42824 is working at 3500 dilution for normal
western blot with 2ug of samples.
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…):
Whole cell lysates
Lysis buffer: Urea lysis buffer
Protease inhibitors: PIC EDTA free
Phosphatase inhibitors
Reducing agent : SDS Sample Buffer
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane: 50ug
Positive control
Negative control
5) Percentage of gel: 12% Acrylamide
Type of membrane: Nitrocellulose
Protein transfer verified: Staining of residual Gel
Blocking agent and concentration: 5% BSA or Milk Powder
Blocking time : 1hr
Blocking temperature: Room Temperature
6) Primary antibody (If more than one was used, describe in
“additional notes”) :
Concentration or dilution: 2000-250
Diluent buffer : 5%BSA or Milk Powder
Incubation time: over night
Incubation temperature: 4oC
7) Secondary antibody:
Species: Rabbit
Reacts against: Mouse
Concentration or dilution: 3000-1000
Diluent buffer: 5% BSA or Milk Powder
Incubation time: 1-2 hrs
Incubation temperature: Room Temperature
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer: TBS-tween
Number of washes: after primary (3x5) and after Secondary (3x15)
9) Detection method: By using ECL reagent.
10) How many times have you run this staining? More than 4 times
Do you obtain the same results every time? YES
What steps have you altered to try and optimize the use of this antibody?
By Optimizing blocking steps by BSA and milk powder, amount of samples
from 20-50ug, Primary Antibody dilutions from 2000-250, Secondary
antibody dilution from 3000-1000
Document attachment: Attaching images of your blot is strongly
recommended and can greatly speed up our investigation of your problem.
 Nothing on the images…..
Regards,

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 ab54532. The details you have kindly provided will provide us with vital information for our monitoring of product quality
I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.
Pleas note that ab54532 is not tested for IP and we do not know if it is suitable.
I apologise for the inconvenience and am pleased to offer you a free of charge replacement (also possible with an alternative antibody; unfortunately, we do not have an anti-Cofilin antibody that is tested for IP) or credit note in compensation.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More

Answer

Thank you for contacting us.

ab42824 is tested in western blot with human and rat samples and is fully guaranteed (https://www.abcam.com/index.html?pageconfig=abpromise). The observed ˜30kDa and ˜90kDa bands might show slight non specificity of antibody however because we haven't done any further test on the identity of these bands so I am sorry we are unable to confirm what these bands are.

We can assure you that, these bands will vanish with slight optimization of the protocol e.g. by using higher dilution of antibody or washing of membrane,10 minutes first wash and 3, 5 minutes washes.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Answer

Thank you for your reply.

Neither ab42824 or ab12866 show any cross reactivity with either Destrin (ADF) or phosphor Destrin. As neither of these antibodies has been shown to work on monkey tissue then they are both eligible for our testing discount program, please see the link below:

https://www.abcam.com/index.htmlpageconfig=resource&rid=11998&viapagetrap=collaborationdiscount



If you would like to take advantage of the of the testing discount program or if there is anything else I can help you with, please let me know.

Read More
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Rat Tissue sections (brain sections)
Specification
brain sections
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
No

Dr. Sophie Pezet

Verified customer

Submitted Sep 25 2008

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