Anti-Nuclear Receptor Corepressor NCoR antibody (ab58396)

Thank you for your reply.

I am sorry the results you have observed have not improved with ab24552. From looking at the results you have shared you appear to be getting very similar results, with ab24552 showing a much clearer band at ˜250 kDa than with ab58396. As discussed previously, the band at ˜250 kDa may well be the band that you are expecting. Although the SwissProt reference for the Nuclear Receptor Corepressor NCoR states that isoforms of ˜260 and 270 have been observed of this protein. The antibodies you have used have both been raised against regions of the NCoR protein which are present in both isoforms so if either or both of the proteins are expressed in your samples they would be expected to be detected. I have not been able to find any further information as to which of the isoforms is most prevalent. I would therefore not be able offer a suggestion as to which of the two isoforms it may be that you are detecting.

As the results you are seeing with both antibodies are consistent (both show bands of ˜250 kDa), I would suggests this supports this being a specific band. If you would like to confirm this further you could isolate the protein in the band and perform Mass Spec analysis. Alternatively, if you have access to a construct which can be used to over-express the protein in a cell line this can often prove a useful positive and negative control.

You are seeing a shift of about 20 kDa in the size of the protein on your membrane. This size of shift is not uncommon for proteins of this size. Separation by SDS-PAGE does not give the exact size of proteins. It relies on the proteins being coated in SDS proportional to their size, providing a negative charge proportional to their size. This is not always the case. The degree of SDS binding the protein can be affected by its hydrophobicity, charge, glycosylation, phosphorylation, or simply the amino acid sequence. This can also be different depending on the lysis buffer and running buffers used when running the gel.

I hope this information has been of help. If you would like for me to help optimise the results, if you could possibly provide me with more details of how you are running the experiment now. I have attached our questionnaire for this reason. Could you also detail how the tissue is being processed (which lysis buffer and the running buffers used)?

I look forward to receiving your reply.

Thank you for your reply and I am sorry for the delay in my response.

In answer to your questions, I think the upper band you are seeing may well be the Nuclear Receptor Corepressor NCoR protein. It seems from your blot that there may be some further optimisation required in the transfer of the protein. With such a large target it is vital to get good transfer of higher molecular weigh proteins. This may be improved by using the following tips:

1. Make sure to use a low percentage gel (preferably 8% or less).

2. Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. This also promotes swelling of the gel, allowing large proteins to transfer more easily. If using a PVDF membrane I would suggest removing methanol from the transfer buffer altogether (with PVDF methanol is only needed to activate the membrane before assembling the gel/membrane sandwich).

3. I would suggest trying wet transfer overnight at 4°C instead of semi-dry transfer.

4. Ponceau red can be used to check for efficient transfer of the proteins.

From your membrane I would also suggest reducing the quantity of protein loaded on the gel and the antibody concentration.

I look forward to receiving your reply.

I am sorry for the delay in getting back to you.

I now have confirmation of the protocol used with the Anti-Nuclear Receptor Corepressor NCoR antibody (ab58396). The Western blot presented on the datasheet of ab58396 was performed using 5% milk blocking in TBST (0.05% Tween) for 1 hour at room temperature. The antibody was then incubated with the primary antibody, diluted 1/1000 in 1% milk in 0.05% TBST at 4°C overnight.

I hope this information has been of help. If you would like me to take a further look at your results could you please provide me with the following information:

1. Would you be able to let me know that the ladder used was and what the two main bands correspond to? Are these ˜150 kDa and ˜250 kDa as you have mentioned before?

2. What is the difference between the blot on the left hand side and the right hand side?

3. What samples are you looking at currently (human/mouse, cell/tissue lysate)?

I look forward to receiving your reply.

Thank you for your reply.

Our experience is that some antibodies work much better under certain conditions. For example, the anti-GAPDH antibody ab9385 has been shown to work much more efficiently with BSA used in blocking than milk. This can be viewed from the following link:

https://www.abcam.com/GAPDH-antibody-HRP-Loading-Control-ab9385.html

This is why I suggested that I have a look at the protocol used to see if there were any suggestions I could make in order to reduce the background currently observed. I will contact the lab to see if they have any specific suggestions of how the antibody performs at its best.

It is difficult to make a conclusion about your results so far as I do not know exactly which bands are which on your blot. Would you be able to let me know that the ladder used was and what the two main bands correspond to? Are these ˜150 kDa and ˜250 kDa as you have mentioned before?

What is the difference between the blot on the left hand side and the right hand side?

What samples are you looking at currently?

I will get back to you as soon as I have any additional feedback from the lab.

Thank you for your reply.

I am sorry for the delay in my response. I will reply in English if you don't mind as I am more comfortable discussing scientific content in this language. By all means feel free to reply in Swedish, I will understand.

The Nuclear Receptor Corepressor NCoR protein is referred to as having a predicted molecular weight of 270 for isoform 1 and 258 kDa for isoform 2 (SwissProt reference O75376). As the protein is also known to undergo a number of different post-translational modifications, this size could vary slightly when viewed on a blot.

Let me reassure you, the image presented on the datasheet of ab58396 is not a faked image, it is a Western blot run with MDA-MB-435 cell extract (human breast carcinoma). It is difficult to assess the exact size, (whether it is 250 kDa as you have observed, or 270 kDa as is predicted) as the ladder used to run with the sample does not allow accurate quantification.

Thank you for sharing the results you have obtained with this antibody. It may well be that the band at ˜250 kDa is the Nuclear Receptor Corepressor NCoR and the band at ˜150 kDa is a non-specific binding or the protein (or a degradation product). In order to be able to advise further, I would like to collect a little further information from you in regards to the protocol used and the samples tested.

To this end, would you mind filling in the questionnaire I have attached to this email? Once I have received the completed questionnaire, I will look at the protocol and see if there are any suggestions I can make that may improve the results. In addition to this, could you label the blot you shared with the molecular weight markers?

This information will also allow us to investigate this case internally and initiate additional testing where necessary.

I look forward to receiving your reply.

The western blot image on the datasheet for ab58396 shows that this antibody was tested on human MDA-MB-435 cells. We have not tested this antibody against rat samples in house, however, based on sequence homology this antibody can pick up a band between 50 and 60 kDa in rat in addition to a band at 270 kDa.