Anti-c-Fos (phospho T232) antibody (ab17933)

Thanks for your email.

I don't think the answer is correct. Please check the following link about fos and fosb, none of them has MW 60 kD. We used this antibody in our experiment, and we worry people will ask us the same question we asked you.

http://www.genecards.org/cgi-bin/carddisp.pl?gene=FOS&search=c-fos Size: 380 amino acids; 40695 Da

http://www.genecards.org/cgi-bin/carddisp.pl?gene=FOSB&search=c-fos Size: 338 amino acids; 35928 Da; NC_000019.8 NT_011109.15 NT_086903.1

Even in your company, you sell c-Fos antibody that detect 46kd (http://www.abcam.com/index.html?datasheet=14285&CFID=4871127&CFTOKEN=75793247), and thus we are confusing.

Thanks again for your help

ANSWER:

Thank you for your reply.

Please see the response from the originator of the antibody.

"We are very confident of the specificity of this antibody which had has been thoroughly tested by us (including on c-fos IP sample using Pan antibodies from other vendors). This is in addition to the testing and data provided by a very well respected lab at Harvard (John Blenis lab) in this area of research. One quick experiment for them to do is IP using other vendors' Antibody prior to immunoblotting with ours."

Please do not hesitate to contact if you have any other questions.

I still have a few questions. We are using RIPA buffer for cell lysis and denaturing our samples by heating to 99 C in Laemmli's buffer. In theory we should not have any dimmers left; the samples should be reduced and denatured. What does this lysis buffer provide that we do not have? If we use this lysis buffer can we run our samples on a standard SDS polyacrylamide gel or does the high salt concentration require different a different gel or removal of the salt before we run the sample?

I thank you for your help

ANSWER:

Thank you for your reply.

The suggested buffer is a modified RIPA buffer. I would agree that you should theoretically not be seeing dimers, but the originator mentioned that they had seen the same problem with other researchers who were using a buffer other than the one suggested.

I was able to get a full protocol for this antibody, which I have included below. I would recommend trying this protocol. If the antibody is still not detecting the 60 kDa band, I would be willing at that point to give you a free-of-charge replacement or a refund.

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

Western Blotting Procedure 1. Lyse approximately 107 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. 2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifuged at 100,000 x g for 30 minutes for greater clarification. 3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates. 4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100oC. 5. Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE. 6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used. 7. Soak the PVDF membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes. 8. Assemble the gel and membrane into the sandwich apparatus. 9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature. 10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes. 11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4oC. 12. Incubate the blocked blot with primary antibody at a 1:1000 dilution in Tris buffered saline supplemented with 3% non-fat dried milk and 0.1% Tween 20 for one hour at room temperature or overnight at 4oC. 13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20. 14. Detect the antibody band using an appropriate secondary antibody in conjunction with your chemiluminescence reagents and instrumentation.

Cell Lysis Buffer Formulation: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 µg/mL pepstatin (alternatively, a commercially available protease inhibitor cocktail may be used)

Transfer Buffer Formulation: 2.4 gm Tris base 14.2 gm glycine 200 mL methanol Q.S. to 1 liter, then add 1 mL 10% SDS. Cool to 4oC prior to use.

Tris Buffered Saline Formulation: 20 mM Tris-HCl, pH 7.4 0.9% NaCl

Blocking Buffer Formulation: 100 mL Tris buffered saline 3 gm non-fat dried milk 0.1 mL Tween 20

I am seeing only a 100kDa band on my Western, and no band at 60kDa. I see that there is a 100kDa band present on the image on the datasheet. Do you know what this protein is?

ANSWER:

Thank you for your enquiry.

c-Fos dimerizes with c-Jun (~40kDa), so this is what the band around 100 kDa may be.

One consideration is why no band was observed at 60 kDa. I have obtained the Cell Lysis buffer recipe from the originator of the antibody. They stated that they have received similar complaints where the researcher used another lysis buffer which was more mild, and caused a similar situation.

I would suggest using the lysis buffer formulation I have added below to see if this improves your results. Please let me know if this information helps.

Cell Lysis Buffer Formulation: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 ?g/mL aprotinin 10 ?g/mL leupeptin 1 ?g/mL pepstatin (alternatively, a commercially available protease inhibitor cocktail may be used)

I notice that you have phospho c-fos antibodies available. Have these antibodies been used for immunofluorescence in tissues?

ANSWER:

Thank you for your enquiry. Ab27793 has not yet been tested for application in IF (only Western blotting so far)but ab17933 - Rabbit polyclonal to c-Fos (phospho T232) - has been tested in IF. There is an image on the online datasheet with more more information.

Please contact us again if you have any additional questions.

What size band is detected with this antibody in Western blotting. It states "Predicted molecular weight: 41 kDa" but the image shows a band running between 50 and 75 kDa.

Also, what is the higher band on that blot (at approx 100 kDa)?

ANSWER:

Thank you for your enquiry and patience. I contacted our source for this antibody and ab17933 detects a band at approximately 60 kDa. c-Fos is an approximately 60 kDa immediate early gene product.

Regarding the Western blot image with the peptide competition experiment data, the originator is not sure what the second band represents. "It's possible that there was a larger protein in the sample that shares a common sequence with c-Fos and was therefore detected in the blot.

The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human c-Fos that contains threonine 232. A BLAST search shows that the sequence of AAs 228-235 around the pT232 bears homology with many, many other proteins. Interestingly, this exact sequence is found in Mouse FBJ osteosarcoma oncogene. According to the data sheets, the peptide competition experiment was conducted using lysates from SKBR3 cells; I wonder, then, if that other band corresponds to some homology with a human oncogene."

If you have any additional questions, please contact us again.