Multiple antibodies give no signal - (Feb/10/2010 )

Hi, I am doing some western blotting on punches of neural tissue that has been homogenized in a Tris-Sucrose buffer with protease and phosphatase inhibitors. We have several proteins of interest, and so are probing with many different antibodies.

Our problem is that some antibodies yield very nice results, yet others yield no signal at all. This is perplexing since we are using the same membranes, same blocking procedure, same incubation procedure, same developer, same secondary antibody, same film, etc. Everything is identical except for the primary antibody. All of our antibodies are rabbit, and our secondary is a goat anti-rabbit. We are using the recommended manufacturer's concentration (1:1000) on all primary antibodies.


Things we have tried to fix the problem:
1. I have tried using new antibodies and buying the same antibody from different companies, but to no avail.
2. I have also tried using longer development times on the antibodies that dont work, but that has not helped either. We still get absolutely no bands on our film.
3. We have tried different blocking methods (methanol then dry, 1hr in 5% BSA), but this does not help.

The problem does not seem to be related to the weight of the protein of interest, as we are able to see 17 and 42 kD proteins but not 11, 17, and 32 kD proteins. It does not appear to be due to phosphorylation status, as we can see one phosphorylated protein but not another. It does not appear to be due to comparmentalization of the protein (i.e., nuclear vs. cytoplasmic), as we can see both nuclear and cytoplasmic proteins but also fail to see both. For instance, I get results with an antibody for total histone H3, but not for total histone H4.

Any thoughts?? I am dying here.

Antibodies were probably created using different immunogen and behave differently. You are lucky in that you have binding with some of the abs. Why can you not just focus on those...providing the source is reliable and there is no lot to lot variation.

Just because a supplier indicates that their ab is specific does not mean it will work in your particular application at the particular concentration you are examining.

You may wish to inquire as to the sensitivity of the abs you purchased.

Your problem is not in the blocking part of the experiment. You also indicated that you tried longer development times. It is not a problem with the 2ndary ab color development...it is with the primary. You also have no background problems indicted in your posting. Why not incubate overnight with the primary and see what happens...could be sensitivity issue.

sgt4boston on Feb 10 2010, 01:52 PM said:

We are using the results of the antibodies that work, but the ones that dont work are the ones we are really more interested in. So, we would really like to get those up and running.

We always incubate overnight. We use a methanol + dry block prior to incubation, as recommended by the PVDF manufacturer, and never have any background issues. We either get the specific band we are looking for or we get nothing at all. It may be a sensitivity issue, but if so I would expect to see at least a very faint band after overnight incubation and hours of development. I am really thinking that the primary is simply not binding to the protein for some reason. It shouldnt be a protein concentration issue since we can see proteins that should be present in the same quantity as proteins that we cant see (in the case of histone H3 and H4).

are you sure that all of your antibodies are validated for western blots?

are against the right species?

have you tried more concentrated than recommended?

some of the primaries may require altered binding conditions (eg- higher or lower salt, etc), what is recommended by the company?

mdfenko on Feb 10 2010, 03:55 PM said:

All of our primary ABs are recommended for Westerns and have been validated on westerns. All are listed as have reactivity with rat tissue (which we are using). We have not tried to increase the concentration because most of these ABs come in 100uL vials, and we use 10 per blot. If we increase the concentration much more, we will be using a whole lot of very expensive AB on one blot.

I will check into the recommendations of the company, but most seem pretty basic.

vezz77 on Feb 10 2010, 02:08 PM said:

100ul vials are very common in the antibody world. You should be able to use less than 10 ml/blot for the antibody steps unless your membrane is huge. I have used 1.0 ml for probing a 8 x 8 cm blot before, and you could probably get away with less volume than that. You need to bag the membrane in a small pouch (e.g. ziplock bag, though I use a heat sealer to create custom made pouches), and then add the antibody dilution to the pouch.

bob1 on Feb 10 2010, 05:45 PM said:

We normally use 10uL of antibody in 10ml of 5% BSA+TBST. That makes a 1:1000 dilution, which is what the manufacturer recommends for westerns. We put it in a custom sealed bag, as you said. If we were seeing very faint bands, I may be tempted to increase the concentration to 1:500, but since we are seeing nothing I am worried that may just be a waste of valuable antibody.

Yeah, you can use small volumes for your membranes.

With the antibodies very often is a lottery, some of them are perfect, some of them are really crap, and normally you just have to test them to know!And in the datasheet all of them work and all of them are tested, but when you try them some of them give you crap... So I would test other companies (if possible) and I would increase the concentration of the antibodies; it is better to spend the antibodies and get a signal that save them and get no signal at all, right? .

Other thing you can try is dilute them in TTBS with 5% milk instead of BSA (or the other way around)

laurequillo on Feb 10 2010, 11:04 PM said:

We have tested another company for two of the antibodies that are failing, and they are not working either. I am more inclined to think we are doing something wrong since these antibodies have been used elsewhere in the literature. I will try to find out if where our protocol differs from the published protocols.

Does anyone think it may help if I blocked in BSA instead of doing the methanol block? Or perhaps if I did both (methanol, dry, then BSA for an hour)?

vezz77 on Feb 11 2010, 03:31 PM said:

Normally after the transfer I use TTBS+5% milk to block. Sometimes I use TTBS+5% BSA but I never use methanol for blocking (I just use methanol before the transfer to "activate" the membrane)