Western Blot - wrong size bands

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Western Blot - wrong size bands

Postby newbie17425 » Feb 15 2017 9:19 am

Hi,

I'm running a Western Blot with rat Hippocampus samples and a Sigma TLR 7 antibody (http://www.sigmaaldrich.com/catalog/pro ... &region=DE). The problem is I didn't get the band at 121 Kda but at 50 Kda (as shown on the Sigma Website).

What could be wrong? Could the ladder be wrong or not representing the correct size or something wrong with the samples?

Thanks.
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Re: Western Blot - wrong size bands

Postby relaxin » Feb 15 2017 12:11 pm

If you look at the Sigma figure, there is a blue arrow point at a specific 120.9 kDa band for TLR7. The figure legend did not address the 50 kDa band. It could be a nonspecific band cross-reacting with the antibody.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 15 2017 1:05 pm

relaxin wrote:If you look at the Sigma figure, there is a blue arrow point at a specific 120.9 kDa band for TLR7. The figure legend did not address the 50 kDa band. It could be a nonspecific band cross-reacting with the antibody.


Is there anything I can do about it.
I ran a 12% gel so the bands from 250-100 Kda were closely packed and didn't separate properly like in the picture (http://www.bio-rad.com/en-ch/sku/161037 ... ds-500-mul). Could it be that proteins didn't migrate properly?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 16 2017 9:44 am

the picture at the website doesn't give you information about the gel.

a 12% gel would give better separation of low molecular weight proteins than high.

you can try a lower percent gel or gradient to improve separation of high molecular weight proteins.

the 50kDa band(s) might be a commonly found artifact caused by keratins and reducing agent.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 16 2017 1:51 pm

mdfenko wrote:the picture at the website doesn't give you information about the gel.

a 12% gel would give better separation of low molecular weight proteins than high.

you can try a lower percent gel or gradient to improve separation of high molecular weight proteins.

the 50kDa band(s) might be a commonly found artifact caused by keratins and reducing agent.


Yes, I tried with a 8% gel and the upper molecular bands have separated nicely. I hope this solves the problem and I get the correct bands.

Can you tell me if the protein size should be different in cell lysate and hippocampus tissues?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 16 2017 2:01 pm

newbie17425 wrote:Can you tell me if the protein size should be different in cell lysate and hippocampus tissues?

no. i'm not familiar with the tlr7.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 16 2017 3:23 pm

mdfenko wrote:
newbie17425 wrote:Can you tell me if the protein size should be different in cell lysate and hippocampus tissues?

no. i'm not familiar with the tlr7.


Ok. Do you suggest a high antibody dilution could be a problem for bands appearing at the wrong size?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 7:09 am

high antibody concentration can cause non-specific binding. you can try diluting further.

if it is, however, the artifact i mentioned then you can eliminate it by omitting reducing agent. sometimes fresh reducing agent will also work.

if it's not that artifact (the webpage for the antibody shows banding around 50 kDa) then you may have to put up with the band.

one other thing, if tlr7 is not a monomer then what you are seeing may be subunits.

(sorry if this isn't helpful)
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 7:27 am

mdfenko wrote:high antibody concentration can cause non-specific binding. you can try diluting further.

if it is, however, the artifact i mentioned then you can eliminate it by omitting reducing agent. sometimes fresh reducing agent will also work.

if it's not that artifact (the webpage for the antibody shows banding around 50 kDa) then you may have to put up with the band.

one other thing, if tlr7 is not a monomer then what you are seeing may be subunits.

(sorry if this isn't helpful)


I diluted it to 1:2000 and no bands appear at either 121 or 50 kDa. Could it be that the proteins are not expressed in my model? I used ACTß as loading control and there were bands in all the lanes.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 8:15 am

it's possible. the way to confirm, of course, would be to try a different model.

the problem might also be your wb incubation conditions (temperature, time, etc.). could you give the procedure for us to review?
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 9:02 am

mdfenko wrote:it's possible. the way to confirm, of course, would be to try a different model.

the problem might also be your wb incubation conditions (temperature, time, etc.). could you give the procedure for us to review?


My protocol is as follows:
After running a 8% SDS PAGE, I blot the membrane for 90’ at 90 V. Then I run a Ponceau staining on the membrane for 5-10 min and wash it until the color goes off. I block the membrane for 1 h in 5% milk in TBS-T followed by over night incubation at 4°C with agitation.
The next day I wash the membrane 5 times for 5’ in TBS-T and incubate the membrane for 1-2h in secondary ab. I again wash the membrane 5 times for 5’ in TBS-T followed by 2 washes in TBS for 5’. I incubate the membrane in ECL reagent for 2 min and develop the film.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 9:30 am

it doesn't have to, but, does the band show up with ponceau? have you stained the gel (preferably with silver) after transfer to ensure that the protein transferred efficiently?

do you prepare your antibodies with tbst alone or do you add blocking agent (milk) to the buffer?

can you try longer exposure?

you can rule out non-specific binding of the secondary antibody (to the 50 kDa band) by omitting the primary antibody from the procedure (probably not necessary because it didn't show up when you diluted the primary, just covering all the bases).

can you load more protein on the gel to ensure that there is a detectable amount for the blot?
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 9:36 am

mdfenko wrote:it doesn't have to, but, does the band show up with ponceau? have you stained the gel (preferably with silver) after transfer to ensure that the protein transferred efficiently?

do you prepare your antibodies with tbst alone or do you add blocking agent (milk) to the buffer?

can you try longer exposure?

you can rule out non-specific binding of the secondary antibody (to the 50 kDa band) by omitting the primary antibody from the procedure (probably not necessary because it didn't show up when you diluted the primary, just covering all the bases).

can you load more protein on the gel to ensure that there is a detectable amount for the blot?


Yes, the bands are there while doing Ponceau, but when I do a Coomassie on the gel (after blotting), I still see some bands (proteins) there too.
I prepare my antibody in TBST-milk.
I load 15µg of protein now, do you think loading more would help?
Long exposure with the ECL reagent before developing or while putting the film for developing. I tried 1 h with the film on the membrane, but it didn't work.
Is 90' at 90V sufficient or can I increase the time?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 10:25 am

transfer is never 100%, large proteins are especially difficult to transfer efficiently (if you want to keep smaller proteins on the blot). you may be able to improve the transfer by adding time if it is a "wet" or tank blot. if you are transferring with a semi-dry or dry apparatus then you must stay within the parameters outlined in the manual for the device.

one thing that will allow you to keep small proteins on the blot while increasing large protein efficiency is to blot to a small pore membrane (0.2um or smaller).

if this is a crude extract then you can load more (maybe up to 50ug, depends on the size and thickness of the gel) protein. then you may have a more detectable amount of tlr7 on the blot.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 10:31 am

mdfenko wrote:transfer is never 100%, large proteins are especially difficult to transfer efficiently (if you want to keep smaller proteins on the blot). you may be able to improve the transfer by adding time if it is a "wet" or tank blot. if you are transferring with a semi-dry or dry apparatus then you must stay within the parameters outlined in the manual for the device.


Sorry, it is not clear what you mean. Could you please explain?

one thing that will allow you to keep small proteins on the blot while increasing large protein efficiency is to blot to a small pore membrane (0.2um or smaller).

if this is a crude extract then you can load more (maybe up to 50ug, depends on the size and thickness of the gel) protein. then you may have a more detectable amount of tlr7 on the blot.


With 15µg of proteins and using 1:500 dilution, I could see protein bands, but wrong size. I just ran a Coomassie with the SDS-PAGE to check the quality of the protein with 15µg of samples, I could see bands on the gel at that time too. Do you think I should load 50µg and try?
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