Hi Mel, including a wee bit more detail about your procedure might possibly help, pretty please.

1. there are no circled bands in your picture.

2. there are no MW standards.

3. no information about your band of interest.

4. No info whether this an IP or a column.

5. no info about the resin.

6. no info on the primary

7. no info on the secondary

8. no info about the sample

9. is it tissue, cell lysate, cell sup, etc?

Am I missing something?

Oh yeah. If you are asking a technical question then bloody well include some technical details of what you are trying to accomplish. We are not mind readers!

Pretty please?

This type of smearing on the top can be caused by following reasons.

1. too much of sample loading

2. degradation of some of your proteins

3. non protein impurities in the sample

4. too much of glycerol in the sample buffer

re arrange & re do the experiment with a dilution series of the test samples. still you get this kind of smearing, that means that your proteins either degraded or the sample contains impurities which can drags the proteins of your sample through the gel.

And also recheck the glycerol content in your sample buffer.

The last thing that you can do is make a slow electrophoresis by reducing the amperage or the voltage.

I think you may helpful the above tips

good luck with your experiments!!

Yes indeed, a little more information might help!!

But I fail to see how this can be anything to do with blocking - in the sense that blocking is intended to reduce non-specific antibody binding to the membrane. Clearly you dont have that problem!

I also foail to see how degradation of a protein results in a higher molecular !!

We see this type of non-spefcific with our polyclonal primaries, but secondaries will also do this depending on what the source of the analysed proteins is. Soif you see the same when you omit the primary, and then different secondaries could be assessed to see if the signals reduce or disappear.

I dont think there is a solution to this, apart from trying with a monoclonal primary, but even then, if the epitope is common, you will pick up bands everywhere - even in your markers.

And you will notice that in the vast majority of papers showing western data, only the band of interest is depicted - all the other non-specific stuff is omitted!!

maybe you should try sowing less sample or diluting more antibodies

@Steingrimur Stefansson

Yes, you are missing something. If you click on the picture rather than the embedded preview, you will see that there is a MW ladder (albeit unlabeled) on the left side and circled bands at the top of the image.

Information about your samples and antibodies would be very helpful. For example, aberrant signal such as this can occur when you use mouse tissue with high blood content (heart, liver) and an anti-mouse secondary antibody due to Igg interference.

As everyone has stated previously, more info on what you are looking for and what you want to achieve would help. Also if you've done a coomassie stain or any stain to see what your gel looks like after running will help.

To me, it looks like there's too much high MW proteins in your sample which is leading to the smears and non-specific binding at the top (as previously mentioned you need to check whether it's your primary or secondary that's causing the issue)

However, let me give you another approach you could look at...

If you just want to show your protein of interest is present you could clean up the PAGE and blot by putting the sample through a filter system with a MWCO greater than your protein of interest? An Amicon Ultra centrifugation device should work... Basically what should happen is that your protein should fall through the filter membrane and any proteins of a high MW be retained in filter - eg, say your protein is 60KDa, you use a 100KDa MWCO filter, your protein in theory should flow through filter and anything above 100KDa be retained. It's working contrary to what you would use the filter for as it's normally used to concentrate your sample and retain it but you're doing the opposite. However, you could also use it to concentrate your protein in your sample by selecting a lower MWCO below your protein...

Bear in mind that this procedure introduces an artificial manipulation step - so if you're looking at native expression in say a cell/bacteria, performing this will add bias to your results as you're actively selecting for your protein.

If you have this problem with different primaries and secondaries, is it possible your lysis buffer or SDS or DTT or sample heating is not right? Assuming of course you are running SDS-PAGE.

The picture seems to me as if you are staining more or less all proteins of your sample. Are you sure that your secondary antibody is not directed against the species of your sample and that you are working with different species for sample and primary antibody host?

I have some assumes maybe I can help

Impurities in your sample, sample overload, protein degradation, may be your primary antibody is polyclonal and bind other proteins in your sample.