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with regard to the antibodies that have been tested and guaranteed (especially for ab72428), what rabbit tissues were used, and what was the localization of the staining? using the same tissue will help to at least confirm the reported reactivity. Aside from your own in-house validation, do you have record of publications that use your antibodies? specifically, are there any that use the antibodies that you've suggested in rabbit tissue IHC-P? |
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ANSWER: |
Thank you for your reply. |
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Inquiry: I am interested in purchasing an nNOS antibody for use in immunohistochemistry/immunofluorescence in rabbit paraffin sections. A number of your antibodies in your catalog state they have reactivity with rabbit, and have been documented for use in IHC-P. In particular, I am interested in ab3511 and ab72428. ab6175 and ab1376 meet the criteria, but use a similar antigen to another antibody that I tested and didn't work. |
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ANSWER: |
Thank you for contacting us. |
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BATCH NUMBER 400854 ORDER NUMBER PO-02237 DESCRIPTION OF THE PROBLEM No signal or weak signal. No bands. SAMPLE Total cell lysate from rabbit cardiac myocyte PRIMARY ANTIBODY 1.Goat polyclonal nNos antibody ( ab1376: abcam) 1 to 1000 dilute in 1% skim milk with in TBS buffer and shake slowing at cold room o/n 2.Wash PDVF membrane in TBST buffer for 10, 5, 5, 5, 5 min with gently shaking DETECTION METHOD ECL plus (pierce) POSITIVE AND NEGATIVE CONTROLS USED N/A ANTIBODY STORAGE CONDITIONS -20 oC SAMPLE PREPARATION Total cell lysate in lysis buffer containing 150 mM NaCl, 50 mM Tris•HCl (pH 8.0), 1% Triton X-100, 2 mM EDTA, and protease inhibitors (Complete EGTA-free, Roche Diagnostics) AMOUNT OF PROTEIN LOADED 20 ug ELECTROPHORESIS/GEL CONDITIONS 10% non-reducing Tris-HCl gel TRANSFER AND BLOCKING CONDITIONS 1. transfer of the proteins from the gel to PDVF membrane for 1 hr at 110V 4 oC 2. PDVF membrane in TBS buffer containing 5 % skim milk and shake gently for 30 min SECONDARY ANTIBODY 1. donkey anti-goat HRP (santa cruz biotechnology) 1 to 1000 dilute in 1% skim milk with in TBS buffer and shake slowing at RT for 1.5 hr 2.Wash PDVF membrane in TBST buffer for 10, 5, 5, 5, 5 min with gently shaking HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Increase the primary antibody conc. … doesn’t work |
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ANSWER: |
Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab1376 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support but please bear in mind that this product has not been tested in rabbit species and may not be suitable for use. However, I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only. Can you confirm that the second antibody used has been giving good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. If the above suggestions did not help to improve your results, then I am afraid that this product is indeed not suitable for use with rabbit samples. In any case, can I encourage you to submit an Abreview via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to other researchers. We reward each Abreview with Abpoints which can be used for discounts off future purchases and gifts. If you decide to submit an Abreview, please include a note that you have already spoken to me, so that I will be able to quickly publish your Abreview, as I have already tried troubleshooting with you. To find out more about our Abreview system, please see the following URL: http://www.abcam.com/abreviews Should you require any further information or assistance, please do not hesitate to contact me. |
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Could you please tell me if the AB1376 nNOS antibody is suitable for IF on cultured C2C12 mouse myoblast cells? There seems to be conflicting information in the reference and the 1 enquiry for this antibody. Thanks |
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ANSWER: |
Thank you for your enquiry. The reference you refer to from the data sheet for this antibody (Ab1376) is relating to neuroblastoma cell lines. The C2C12 cell line you are using is a myoblast (skeletal muscle stem cell) cell line. According to a reference I have found, this cell line may require differentiation before it will express nNOS: D. Blottner et al. Cell and Tissue Research 292, 2, p293-302. Nitric oxide synthase in mouse skeletal muscle development and differentiated myoblasts. There is also another reference that may be helpful to you: FEBS Lett. 2000 Sep 29;482(1-2):65-70. Neuronal nitric oxide synthase localizes through multiple structural motifs to the sarcolemma in mouse myotubes. Abdelmoity A et al Although this antibody has been tested in mouse (so will detect mouse nNOS expressed by cells), it has unfortunately not been tested for immunocytochemistry. It has, however, been used for immunofluorescent immunohistochemistry on tissue sections. I hope this helps. Please don't hesitate to get back in touch with me if you require more assistance. Good luck with your experiments!
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the antibody ab1376 for nNOS picks up bands at sizes of 45 and 160kDa. what is the band at 45kDa? is it nNOS as well as the 160kDa band. If it is nNOS why is it smaller than 160 kDa. I have used the antibody and get large bands at 45kDa but very little at 160kDa and would like to know what i am detecting.
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ANSWER: |
Your results are in keeping with those illustrated on our datasheet where we see two bands - a stronger band at 45kDa and another at 160kDa. The larger band is in keeping with the predicted molecular weight of nNOS, but we do not know the precise identity of the smaller band. |
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