Anti-Filamin antibody [FLMN01 (PM6/317)] (ab3261)

I have a question about anti filamin antibody. I'd like to perform the western blotting analysis using the anti-filamin antibody. In filamin there are three isoforms,termed Filamin A,Filamin B and Filamin C. I'm looking for the anti filamin antibody that react to Filamin C. Can your antibodies (ab18121, ab3261 and ab20716) react to Filamin C?

I send this mail from Japan. Thank you for your kind correspondence.

ANSWER:

Thank you for your enquiry.

Regarding ab20716, reactivity to the different isoforms of filamin has not been characterized for this antibody. We have not tested this antibody for reactivity to Filamin C.

I am still looking into the other two antibodies for you.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

i would like to use a filamin antibody for western blot and immunofluorescence. The antibody has to be able to react with human and mouse tissue. Do any of your filamin antibodies, match these criteria?

ANSWER:

Thank you for your enquiry. I'm afraid we do not have anything that exactly matches your requirements but we do have the following which partially match:

ab3261 ab18122 ab11074 ab20485

You can look at the datasheets for these on our website. I hope one of them may be of use to you.

Please feel free to contact me if you have any more questions regarding this.

Where is the epitope located for ab3261? Is it in the N- or C-termial part of filamin A? Filamin A can be cleaved by calpain into a 100kD C-terminal fragment. Can ab3261 detect this fragment in Western blotting? Thank you.

ANSWER:

Thank you for your enquiry. Unfortunately the epitope recognised by ab3221 has not been mapped. The antibody detects the 250 kDa filamin in WB, we have no data concerning the detection of fragments with this antibody, my apologies for the inconvenience.

Please do not hesitate to contact us again if you need further information,

ANTIBODY CODE ab3261 BATCH NUMBER 47457

DESCRIPTION OF THE PROBLEM No staining

SAMPLE I have used mouse embryo sections of various ages. I have also tried skin sections as a control.

PRIMARY ANTIBODY Abcam Filamin antibody at 1:100 followed by 3 5 minute 1xPBS washes. I have tried the primary for 30 minutes, 1 hour or overnight at 4 degrees.

SECONDARY ANTIBODY Vector Laboratories Ltd MOM kit secondary antibody at 1:200 for 45 minutes followed by 3 5 minute 1xPBS washes. When not using the MOM kit I have used Vector Laboratories Ltd Goat anti mouse secondary at 1:200 for 45 minutes

DETECTION METHOD I used SIGMA Streptavidin Cy3, 1:100 diluted in 1xPBS for 30 minutes. I then mounted slides in DAPI-DABCO.

POSITIVE AND NEGATIVE CONTROLS USED I incubated 1 section on each slide in blocking reagent instead of primary antibody

ANTIBODY STORAGE CONDITIONS The antibody has been stored at +4 degrees C since I recieved it.

FIXATION OF SAMPLE I have tried tissue fixed in 4% Paraformaldehyde and tissue fixed in Formalin (both overnight)

ANTIGEN RETRIEVAL I have tried 10mM Citrate buffer pH6 for 10 or 15 minutes. I have also tried Proteinase K and HCl/Borate retrievals.

BLOCKING CONDITIONS Because I am using mouse tissue I have used the Vector Laboratories Ltd Mouse-on-mouse (MOM) kit. This has 2 blocks, 1 for an hour with the mouse IgG blocking reagent and one for 5 minutes with the MOM Diluent. I have also tried doing it without the MOM kit and blocking for 1 hour in 4% Normal goat serum

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 8 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have tried everything

ANSWER:

Thank you for your enquiry and your patience. This antibody has a broad specificity and reacts with several animal species.

Using mouse NIH3T3 cells, this antibody works well in WB with MW of 250 kDa and it has been tested on human skin and works very well.

We would suggest using human skin as a positive control and also testing the secondary antibody and the detection system with another primary antibody.

Should you still have problem with this product or need any help then please do not hesitate to contact us again providing your Purchase Order Number or Abcam Order Number.

Does this antibody react with Filamin A, B, or C? or all of the above?

ANSWER:

It is unknown as to which Filamin type the antibody reacts with.