Anti-OLFM4 antibody (ab105861)

Thank you for your email. I have sent the lysates ab29889 with order number 1007989. I hope this will help.

I have now received a reply from lab. They have confirmed observing a 57kDa using human, rat and mouse liver extracts and 57 kD/ 40 kD band in the stomach extracts. I am OK for sending positive control lysates. However I would be interested in getting your thoughts about the publication that I have sent in my previous email. Looking forward to hearing from you soon.

Thank you for your email. I have forwarded the data to my colleague in lab and now I am waiting a reply. I will contact you again soon; normally they write back to us within 24 hours. I have also done further search which I would be happy in sharing with you. The 57kda molecular weight size is of unmodified protein as given in Swissprot and as per same database the OLFM4 protein is further processed in mature form with deletion of signal peptide or addition of other molecules that leads to full functioning of protein. It may well be that in the cancerous tissues or cell line OLFM4 is further processed in small isoform. Please check the attached publication (Fig1) which is supporting this; in this publication the author has observed 2 mRNA constructs in different tumour tissues size 1.7 kb and size 2.9 kb. http://cancerres.aacrjournals.org/content/64/7/2474/F1.expansion.html http://cancerres.aacrjournals.org/content/64/7/2474.long I have also carefully checked the images you have kindly provided, I am presuming that the cell lines you have used are all cancerous cell line (T3H4, HCT116, FA6 and MiaPaca2 are in fact the cancerous cell lines). Interestingly the CFPAC1 do show a band at 57KDa in addition to 37kDa. I have checked few more publications; unfortunately because cut out images are given so it was hard to understand the actual m.wt. other scientist has been observing. It will be a good of emailing few of them and discussing the results. I will contact you as soon as I receive a reply from lab. Many thanks for having patience.   

Thanks you very much for contacting us. My colleagues has just forwarded your details to me. I am sorry to hear that you are observing multiple bands again. I will certainly look into this case further and would also like to discuss this case with lab. Could you be so kind sending me the image for my records? There are also 5 isoforms given in Aceview database; http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=human&term=olfm4&submit=Go. I am not sure if any of the isoform is reported in the lysates you have been using however because both antibodies gave similar results so it will be worth checking the literature. I will review the product again with data I receive from lab.  Regarding the lysates; I will send these once I finish reviewing this product. Looking forward to hearing from you soon.

Thank you for contacting us.  Below is the IHC protocol for ab105861.  I hope this helps, please let me know if you have any additonal questions.   Tissue Preparation: Formalin fixation and embedding in paraffin wax Tissue Sectioning: Make 4-μm sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60°C. Deparaffinization: Wash dry slides in 3 changes of xylene – 5 minutes each @ RT Rehydration: Wash slides in 3 changes of 100% alcohol – 3 minutes each @ RT Wash slides in 2 changes of 95% alcohol – 3 minutes each @ RT Wash slides in 1 change of 80% alcohol – 3 minutes @ RT Rinse slides in gentle running distilled water – 5 minutes @ RT Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes Remove from heat and let stand at room temperature in buffer - 20 minutes Rinse in 1X TBS with Tween (TBST) – 1 minute @ RT Immunostaining: (Do not allow tissues to dry at any time during the staining procedure) Apply a universal protein block – 20 minutes @ RT Drain protein block from slides, apply diluted primary antibody (5ug/mL) – 45 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply alkaline phosphatase streptavidin – 30 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply alkaline phosphatase chromogen substrate – 30 minutes @ RT Wash slides in distilled water – 1 minute @ RT Dehydrate:  (This method should only be used if the chromogen substrate is alcohol insoluble (e.g. Vector Red, DAB) Wash slides in 2 changes of 80% alcohol – 1 minute each @ RT Wash slides in 2 changes of 95% alcohol – 1 minute each @ RT Wash slides in 3 changes of 100% alcohol – 1 minute each @ RT Wash slides in 3 changes of xylene – 1 minute each @ RT Apply coverslip