Anti-68kDa Neurofilament/NF-L 抗体 (ab9035)

Thank you for your reply.

Trying the antibody in WB is a good alternative to find out whether there might have been a problem with it. For WB, we recommend a 1/5000 dilution, and it should recognise a band at ˜68KDa under denaturing and reducing conditions.

I would encourage you to have a look at our online protocols to perform the assay:

https://www.abcam.com/index.html?pageconfig=resource&rid=11375

Please let me know how the antibody performs in this technique, and I will be more than happy to help you further.

Thank you for your reply.

I can confirm that without controls it is not possible to interpret the results. Also this antibody is not experimentallytested for mouse samples. Although it is very likely that ab9035 works with mouse samples, in this case further control would be good. For example a positive control from rat.

Cell lines often show a peculiar expression pattern of proteins since they are not primary cells and have been cultured for a long time. Cancer cells in particular have expression pattern thatcan vary from what is expected.Therefore, as you mentioned, I suggest to use a positive control and a negative control.

A suitable positive control would be brain or the dorsal root ganglion. Spleen could be the negative control. Please follow this link for more information on the expression pattern of this protein in rat:

http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Rn.18568

I hope this information is helpful and wish you good luck with your experiments.

Thank you for your inquiry.

Unfortunately, I am unable to provide a straight forward answer to your question. Since you are treating cells for neuronal differentiation and I assume that the untreated cells are supposed to be negative for the 68kDa Neurofilament, the untreated cells could be one negative control. I suggest to use a cell type as positive control that does not need treatment.

What cells are used? Rat or pig?

To interpret the results of any experiment, multiple controls are required:

Positive control

A section from a cell line or tissue known to express the protein you are detecting. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.

We recommend to check the antibody datasheet, which will often provide a suggested positive control. Always ensure the tissue or cell line you use is from a tested species. Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances:
Check to see if there are any Abreviews for the antibody. Any tissues, cells or lysates that have been used successfully by these customers can be considered a suitable positive control.
Try looking at the http://expasy.org/sprot/ or http://omnigene.sourceforge.net/index.shtml database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls.
Check the http://www.genecards.org/ entry for the protein. This will usually provide you with relative levels of expression in various tissues.
If you still have difficulty finding a suitable control, we recommend doing a quick literature search on http://www.ncbi.nlm.nih.gov/sites/entrez to see which tissues and cells express the protein of interest.

Negative control (if possible)

A section from a cell line or tissue sample known not to express the protein you are detecting. This is to check for non specific binding and false positive results.

No primary control

This is when the primary antibody is not added to one strip of membrane. Secondary antibody only is added. This indicates if any non specific binding or false-positivesmay be due to non specific binding ofthe secondary antibody. Antibody dilution buffer containing no antibody is used in place of the primary antibody solution at this point in the procedure. The secondary antibody is incubated on the sample in the same way as usual.

Isotype control

Instead of the primary antibody, aantibody of the same isotype without any specific binding capacities is used. This will serve as the background control for the specific primary antibody. We suggest to use this control when adjusting the instrument to take images of the ICC or IHC.

If background occurs in the isotype control, the gain can be decreased until this background is gone. These setting then can be used to evaluate the staining of the specific antibody. All positive staining can now be assumed to be specific.

I am sorry that I could not provide a straight forward answer on this occasion and hope this information is helpful.

Thanks for your inquiry. I checked with the lab and this is what they had to say: "The sample was serum, but in that particular batch there was some hemolysis resulting in release of some hemoglobin from red blood cells. We try to avoid shipping material like that to you as it is a little ugly looking. We have tested the serum and the presence of the hemoglobin has absolutely no impact on the efficacy of the antibody." I hope this was helpful. Please feel free to contact me if you require further assistance.