How did other signals look like. Did you get sharp bands there?

The fusion of bands as well as their diffuse shape could be due to the loading of 30 µg. In my experience this is already a lot and you should be carefull that your antibody signals are still within the linear phase for quantification.

Dear Tanmay,

I have had this happening sometimes as well. There are a couple of reasons that this could be happening:

-Loading too much protein could cause this although this might not be the case in your experiment. Still, you could try slightly less protein if your bands of interest are sufficiently visible.

-Samples can diffuse if the loading is slow. This could be visible with naked eye, if you see your dye diffusing too while loading. Are your other bands diffused too? Answering that question can give you an idea if there is a general problem in your runs or if its beta actin specific

-It could be a matter of ionic strength, you could try increasing salt concentrations (TrisCl, NaCl). More on this can be found in the link provided.

Some extra ideas to tackle it would be:

-Try a shorter incubation of your secondary (1h is the standard in our lab) or try an even more diluted secondary ( i would go for 1:5000 and see how this goes)

-Are you using films for the exposure/development? You could try shorter exposure times, while it might not solve the problem it could help you defining bands and subtracting backgroung during densitometry.

-You could also try an initial quicker run during the SDS-PAGE phase. Instead of 50v start with 80v and see if this gives you well defined bands.

https://www.researchgate.net/post/Proteins_bands_in_western_blot_are_merged_does_any_one_know_why

I usually load about 10 ug of protein lysate in order to obtain the best western possible. Try to reduce your loading. 10 ug is quite enough (unless your protein of interest is in low amount in the cells so you really need that much of loading material).

Hello

This problem is occurring during the running of the gel (stain one, you'll see). if you look closely, you will see that the erk also has connections between lanes, albeit not as strongly stained as with actin.

A few factors that can cause this:

sample load- there may be too much protein

time between loading and start of the run- diffusion of the sample will occur

salt in the sample buffer- salts can have strange, unpredictable effects

other additives in the sample buffer (eg detergents, lipids, peg, organics, etc)- (see salt)

gel type- gradients are more prone to this, especially when run slowly you will see more sideways diffusion of the bands, more so with smaller proteins (path of least resistance)

gel concentration (including %c)- restricted mobility

Beta actin is a housekeeping protein used to see (in most cases) as loading control in your gel. It is present in good amount inside the cells, therefore you are getting diffused bands while loading 30 ug protein....u can do the following things:

1. load proteins in a range of 5 ug, 10ug, 15 ug, 20ug and see which amount gives ou sharp signal.

2. May be while loading protein samples into the wells of PAGE, you are taking little bit more time in loading and to start running your gel..in this way the protein starts diffusing horizontaly and this results into diffused bands.

3. Make sure that before putting ECL (detecting substrate) over your membrane then the membrane should be dried...if it is wet then the signals would come as diffused.

4. Check the expiry of your developing films.

5. for beta actin..no need to keep for overnight primary antibody incubation...you can keep it for few hours at room temperature and it will works well.

6. If u want to keep for overnight than increase the dilution of the antibody...use 1:5000.

Cya

Hello Daniel & Theofanis,

bands for other proteins are fine they are not fused. Only that beta actin giving me trouble.

Thanks!!

-After Putting ECL mixture over the PVDF membrane we place membrane in plastic sheet and take images with the help of Gel Logic Pro 2200.

-I cant see any dye is diffusing sideways while running the gels.

-Next time I am going to run gels at 80V. will try with lesser concentration of sec antibody(1:5000). Lets see what I get. Otherwise I have reduce the loading concentration and try.

Thanks a lot guys!!

It seems as loading and gel running problems.