12 replies to this topic

[myxobacteria]

Hello to everybody. It is a pleasure joining such a marvellous community!

I have a persisting problem with my western-blots which is really annoying me. I always obtain a smear in the lines of my westerns and I do not know where they come from. It starts in the wells and go down until the middle of the membrane, approximately. I have tried several variations:

1. I tried two different buffers to isolate proteins, one contains SDS and DTT, while the other contains glycin, triton and beta-mercapto.
2. I have varied the quantity of proteins loaded, from 10 to 50 micrograms. I always get the same smear.
3. I also varied the concentration of first antibody (from 1:2000 to 1:20000) and the stringency of washes

With all these variations I could observed difference in the band pattern (I mean, below the smear), but the smear is always the same. The only idea I have is that maybe I have a conamination with glucids, but I do not know whether this is possible and, if so, what can be done.

May anyone help me, please?

[science noob]

myxobacteria, on 13 December 2011 - 04:03 AM, said:

Hello to everybody. It is a pleasure joining such a marvellous community!

I have a persisting problem with my western-blots which is really annoying me. I always obtain a smear in the lines of my westerns and I do not know where they come from. It starts in the wells and go down until the middle of the membrane, approximately. I have tried several variations:

1. I tried two different buffers to isolate proteins, one contains SDS and DTT, while the other contains glycin, triton and beta-mercapto.
2. I have varied the quantity of proteins loaded, from 10 to 50 micrograms. I always get the same smear.
3. I also varied the concentration of first antibody (from 1:2000 to 1:20000) and the stringency of washes

With all these variations I could observed difference in the band pattern (I mean, below the smear), but the smear is always the same. The only idea I have is that maybe I have a conamination with glucids, but I do not know whether this is possible and, if so, what can be done.

May anyone help me, please?

Smearing would most likely be caused during the SDS-PAGE step. Could be an indicator that total protein wasn't separated evenly? What are your SDS-PAGE settings?

[myxobacteria]

I use a 12% 30:0.8 acrilamide:bis gel run for 1.5 - 2 hours at 120 V.

Anyway, I checked protein isolation on a normal comassie stained gel and it looked pretty well.

[Biouday]

myxobacteria, on 13 December 2011 - 06:37 AM, said:

I use a 12% 30:0.8 acrilamide:bis gel run for 1.5 - 2 hours at 120 V.

Anyway, I checked protein isolation on a normal comassie stained gel and it looked pretty well.

Look like your protein is degrading. even you see protein in normal comassie stained gel, you cannot really see a degraded or degrading protein there. But antibody can detect it.

Can you tell what is the source of the protein.

How you are storing your protein

Did you boil the protein solution enough time.

If possible upload both comassie stained gel and your western blot result that help other to understand your problem quit easy.

[myxobacteria]

It is a total protein extract from Aspergillus. I would like to check the normal expression level of a tagged-protein under natural conditions.

I thought of degradation, however, is it not strange that the smear appears in the wells, and not at the bottom of the line?

I store the protein solution directly at -20°C. It is not long since I isolated them, just three weeks.

I boil proteins 10 minutes in a thermo-bloc at 100°C

Here they are, the comassie and the last western:    http://imageshack.us...4/comassie.jpg/

By the way, the band in the last line is not the good one, before you think so, since that is exactly the negative control line (extract without tagged-protein), and also the size is not correct.

[science noob]

myxobacteria, on 14 December 2011 - 01:49 AM, said:

It is a total protein extract from Aspergillus. I would like to check the normal expression level of a tagged-protein under natural conditions.

I thought of degradation, however, is it not strange that the smear appears in the wells, and not at the bottom of the line?

I store the protein solution directly at -20°C. It is not long since I isolated them, just three weeks.

I boil proteins 10 minutes in a thermo-bloc at 100°C

Here they are, the comassie and the last western: http://imageshack.us...4/comassie.jpg/

By the way, the band in the last line is not the good one, before you think so, since that is exactly the negative control line (extract without tagged-protein), and also the size is not correct.

What is the size of your protein of interest? And how much total protein did you load in the well?

[myxobacteria]

The size I expect is 32 kDa. I think there is a very soft band in one of the lines...

In the comassie gel I loaded 35 micrograms. In this particular western 50 micrograms (I have also tried 10, 20 and 40)

Thank you for your interest and help, by the way

[science noob]

myxobacteria, on 14 December 2011 - 03:09 AM, said:

Thank you for your interest and help, by the way

I've been doing alot of westerns recently and haven't had much experience prior to this so I really think this forum is useful in sharing and troubleshooting.
How do the 10, 20 and 40 look vs 50 ug?

There seems to be a faint band around the 32kDa size (in the forth lane sample from left and last lane) - your band should appear in between the 3rd and 4th last ladder (25-35 kDa region).  But I seriously haven't seen such a smear before.  Try reducing the voltage of your SDS-PAGE to maybe 90 or 100V.  Did you try to stain for a housekeeping protein (e.g. actin, tubulin, GAPDH)? Might be worth doing that to see if those are even present.

If your protein of interest isn't a large one (falling in the bad smear region), I would not worry as much because the bottom half is pretty clean and any potential band should turn up.

[myxobacteria]

In the 10, 20 and 40 ug westerns the smear looked the same.

I am going to use a E. coli extract with a protein containing the same tag overexpressed to check whether the smear appears and also whether I can clearly detect an overexpressed protein. I´ll try less voltage, following your piece of advise.

I really hope to discover what is going on there...

Thank you again

[mdfenko]

that "smearing" may be due to aggregation of your protein

[myxobacteria]

mdfenko, on 14 December 2011 - 12:54 PM, said:

that "smearing" may be due to aggregation of your protein

To be honest, I don´t think so, since I am expecting a very very low expression level. However, it is worthy considering all possibilities. May you tell me how can I avoid aggregation, please?

[mdfenko]

depends on the protein. some aggregate readily and require some additives to prevent aggregation.

you may also be boiling the sample in sds buffer to little or too much. you can reduce the temperature of the sample heating to about 65C and incubate for 10-20 minutes.

[myxobacteria]

Ok! I will try your piece of advise. Thank you very much