Very simple: buy a cheap secondary from obscure vendor. You will have to buy a secondary that has minimal cross-reactivity to major serum proteins in other species. I would cut your blot in half, since the top half will have your 150kDa band and the bottom half the 42+50kDa bands. Then label the two halves with the one antibody and the appropriate secondary accordingly.

Before jumping to any conclusions, first let's see if your secondaries are playing you any tricks.Have you compared with and without primary? Do you keep the blots for the mouse antibody separate from the blots for the goat antibody?

It is also important to address the integrity of your antibodies. Make sure they do what they are supposed to do before combining anything in a multiple labelling experiment.

i detect both proteins on the same blot. detect synaptopodin first, washing step, then detect actin. but it is possible come to think of it but how on earth can secondary anti mouse react with goat primary anti antibody?

Very simple: buy a cheap secondary from obscure vendor. You will have to buy a secondary that has minimal cross-reactivity to major serum proteins in other species. I would cut your blot in half, since the top half will have your 150kDa band and the bottom half the 42+50kDa bands. Then label the two halves with the one antibody and the appropriate secondary accordingly.

Ive been thinking it over time but i dont really get your idea. If i get your suggestion right, it is not so likely that 2 secondary antibodies (one against primary goat synaptopodin    and      one against    primary mouse actin) binds to the 150kd portion of some random protein. 

Cross-reactivity of primary or secondary antibodies can be a problem in western blot analyses. When samples run on the same gel, I often cut the blot and incubate sections with different antibodies, as appropriate, to avoid/check issues such as cross-reactivity or when different blotting conditions are required.

Iwan,  I would listen to what Jan has to say, I think he has hit the nail on the head.  Unless your antibodies have been pre-adsorbed you can expect, or should expect cross-reactivity.

Hmm first i incubated the blot (heres only 1 lane) with primary antibody(goat source) detecting synaptopodin and then the anti goat sec antibody. Then i detect by exposure.

After this i washed it 3x with tbst for 5 mins each. Incubate with anti actin primary antibody(mouse derived) followed by incubation with anti mouse secondary antibody then detect actin by exposure. 

If cross reactivity(between any of the 4 types antibodies mentioned) is an issue , why do i see the 150kd band from first detection (even before anti actin and the secondary antibody)? Cross reactivity as in the primary antibody against synaptopodin binds non specifically to 150kd protein OR secondary antibody binds to 150kd band?

Indeed, and one way to find out is to compare with and without primary.

Are you reducing your samples along with boiling for 10 min?

Once again Jan is on the money.  Really that should be the first step.  You could run the samples reduced and non-reduced.  Be sure to alkylate the samples after reduction.

1. Make sure you are using the right lysis buffer to prepare your samples.

2. Check your blocking buffer and the buffer you use to dilute your secondary antibodies (if you use milk there may be some non specific bands, I use 10% BSA in TBS for primary and secondary which again depends on the protein of interest)

3. If you detect your protein bands by chemiluminescence and have issues due to potential cross reactivity then try using fluorescent labeled secondary antibodies from Rockland. That way you may be able to eliminate cross reactivity issues. 

I think the answer is much easier...you are not stripping your blot between exposures!! Of course the signal you see in the first exposure remains there, you are just not washing out the first synaptopodin primary+secondary antibody (If 3x 5min TBST would be enough to strip antibodies, nobody would detect anything in western blot), and then you get the same signal (fainter, because you washed a little bit more) plus an additional signal of the actin. If you want to make sure this is the problem, do it the other way around, detect the actin first and then the synaptopodin, I bet you will find the two bands only after synaptopodin detection (the last detection). In case you are not convinced that the 150 KDa band is your protein of interest, knock it down by siRNA  and check the amount you find. Complementary, you could also overexpress it and check if the amount increases. I think these are the more simple tests for antibody specificity.

Please check out what Victor just mentioned. If this is the reason (what I think too), this could speed up the trouble shooting.

i think i get it now but how would one explain the 150kd band seen from first exposure involving primary anti-synaptopdoin and its corresponding secondary antibody only? it is 50kd above the expected size. Is it due to maybe secondary antibody binding with non specific proteins in the blot?

note: this exposure precedes anything before addition of primary actin antibody and its corresponding secondary antibody

Well, there are several possibilities for the 150 KDa band. First, 50 KDa looks like a lot, so we can probably discard post-translational modifications, although heavily phosphorylated proteins can have strong shifts in mobility. Then, one explanation could be the cross-reactivity of the secondary antibody. To check that, just leave out your primary with the same protocol. Other possibility is the cross-reactivity of the primary itself. Even monoclonal primary antibodies give very frequently unspecific bands. This may even change from batch to batch, or depending on the storage (uncorrectly stored antibodies, or very old ones can start detecting additional bands, even completely stop working on the original target) If by any reason your protein is not very abundant in the cell line /tissue you are using, you might be detecting preferentially an unespecific band. Last, perhaps the antibody you are using simply just doesn't work. You would be amazed of how many monoclonal antibodies out there detect incorrect targets, and simply don't work for the intended target. I recommend you to do several things (which in my opinion should always be done when using a primary antibody we have never used before) First, check the literature. Try to find publications where people detect your protein of interest, preferentially in the same tissue/cell line of your interest, and order the antibody that is well referenced. If,as it is sometimes the case, there is not well referenced antibody for your protein, then I would try to go for abcam, cell signalling or millipore antibodies. It is my experience that these three companies usually offer reliable antibodies, although there are always exceptions. In any case, I have good experiences with the customer service of the three companies, so if you show them that the antibody (or the batch you got) doesn't work, they will send you a new batch for free without too much of a problem. Then, once you get your anitbody, validate it. This is a very important step, I would never trust an antibody I have not validated myself. For that, as I said before, order an siRNA (or a couple of them, just in case one of them doesn't work, or, as before for the antibodies, choose one that has been previously used in the literature) and make just a preliminar experiment transfecting a cell line that express your protein with the siRNA against your protein, and the same cell line with a control siRNA, and use these lysates to check the specifity of your antibody. It looks like a pain, and perhaps it is, but I can tell you that in the long run, doing this routinely with your antibodies will save you time, since you will only make your real experiments with an antibody you know works. Although you loose three days in the validation, then you will be able to get meaningful results right away after that, and you won't spend part of the valuable samples, your time and money using an antibody that doesn't work. I hope this advice is helpful!!

If you would have checked the antibody specificity against Synaptopodin and acting you would have found that they cross match, i.e. to say in simple terms, that the antibody would bind to both the proteins. Thats where lies your confusion....

Will strongly recommend, after you trouble shoot most of the above and still unable to figure things out, you match membrane bands with replicate gel and submit excised bands for mass spec ID. This will give definitive identity of your 150K/50K bands.