Western Blot - wrong size bands

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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 24 2017 12:54 pm

newbie17425 wrote:This was the 1st time I used TLR 7. I'm going to repeat it on Monday.

I will try to clean the vessels before incubation steps on Monday. Should I wash the vessel after each step i.e. blocking, pri. antibody?

it shouldn't be necessary to clean it more than once for each use (prior to or after each use) as long as you perform each step, including washes, in the same vessel. more important is to ensure that the membrane moves freely in the vessel with the solutions. prevent the membrane from sticking to the vessel.

some blotches are caused by aggregated proteins (antibodies, block, etc). you should make sure that there is no sediment in the blocking and antibody solutions.

good luck
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 24 2017 1:30 pm

Will decreasing the antibody concentration help decrease the background?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 24 2017 1:49 pm

newbie17425 wrote:Will decreasing the antibody concentration help decrease the background?

it could. but you're already having detection problems. you may want to try that if the other modifications don't eliminate the background.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 25 2017 6:33 pm

Thanks. I will try as we discussed. Out of curiosity, does primary ab incubation at 1 h at RT also works?

I also wanted to mention something about my sample preparation. I prepare samples in Laemmli buffer in 4:1 ratio, heat at 95°C, make aliquots and store at -20°C. Before loading, I again heat the sample and then load. Should I avoid heating the samples again? Also, for how long can I store the samples at RT in sample buffer?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 27 2017 12:17 pm

newbie17425 wrote:Thanks. I will try as we discussed. Out of curiosity, does primary ab incubation at 1 h at RT also works?

it depends on the avidity of the antibody. when i first started, i incubated for 3 hours but ultimately found that 1 hour was sufficient for my antibody.
I also wanted to mention something about my sample preparation. I prepare samples in Laemmli buffer in 4:1 ratio, heat at 95°C, make aliquots and store at -20°C. Before loading, I again heat the sample and then load. Should I avoid heating the samples again? Also, for how long can I store the samples at RT in sample buffer?

i rarely store samples but i find that some people do and some don't. the only thing that i would caution you about is to ensure that all of the sds is back in solution (it crystallizes at low temperature).

as for rt storage, once the protein is denatured it shouldn't degrade (unless a sds insensitive protease is present). so, storage at rt can be indefinite. however, in general, good practice is to use samples as soon as possible or store them frozen.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 28 2017 11:32 am

So, I ran the samples with 25µg of proteins, the background was less as compared to the last time. However, in one sample the background is too high. Do you think my samples have gone bad? What can be done to improve the sample quality?

Thanks.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 28 2017 12:25 pm

if it's just one sample then the likelihood is that the background is being caused by aggregation of the protein in your sample (including your protein of interest). this can cause streaking, smearing, spotting, etc.

this can happen by under or over boiling the sample. incubation at 100C, in the presence of sds and 2me, should not be for more than 5 minutes or proteins may aggregate.

a safe way to "boil" the sample is to incubate at 60-70C for 10-20 minutes. denaturing should be complete and the proteins should not aggregate.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 28 2017 4:23 pm

mdfenko wrote:if it's just one sample then the likelihood is that the background is being caused by aggregation of the protein in your sample (including your protein of interest). this can cause streaking, smearing, spotting, etc.


this can happen by under or over boiling the sample. incubation at 100C, in the presence of sds and 2me, should not be for more than 5 minutes or proteins may aggregate.

I suspect it to be smearing. This could have happened as after boiling I aliquot the samples at -20°C, and the day I use the samples, I boil again for 5 min at 95°C. I think I should avoid repeated boiling. What do you suggest? Also, as the samples are at -20°C, can I keep them at RT 1 day before or can I just put them at 37°C incubator for faster thawing (someone suggested this from a different lab).

mdfenko wrote: a safe way to "boil" the sample is to incubate at 60-70C for 10-20 minutes. denaturing should be complete and the proteins should not aggregate.

Do I need to boil the sample buffer once it is prepared? I prepare my sample buffer with DTT, I prepare 2 ml, boil it at 95°C and then add it to the samples and I boil the sample once I add the sample buffer.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 28 2017 5:17 pm

you can leave the sample at rt overnight or thaw at 37C, your choice. either way, make sure that all of the sds is back in solution or you will experience streaking, etc.

i've never boiled the sample buffer after preparation (this is the first time i'm hearing of the practice), only after adding to the sample. the reducing agent will oxidize, at least partially, and render the sample buffer less effective.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 28 2017 5:30 pm

mdfenko wrote: i've never boiled the sample buffer after preparation (this is the first time i'm hearing of the practice), only after adding to the sample. the reducing agent will oxidize, at least partially, and render the sample buffer less effective.


Maybe this could be a reason as to why I'm seeing streaking/aggregation of proteins. I will prepare fresh samples tomorrow with fresh sample buffer and see how it works.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Mar 01 2017 4:39 pm

Also, all my proteins are not getting transferred to the membrane. After transfer I run a Coomassie on the gel, and I still see some bands around 200-250 kDa. I add 20% methanol to my blotting/transfer buffer. What can be done for efficient transfer?

Also my ECL films are getting bit darker after development. How can I correct this. There is no other source of light except the red light in the dark room.

Thanks.
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Re: Western Blot - wrong size bands

Postby mdfenko » Mar 02 2017 3:04 pm

it's normal to incompletely transfer the protein, especially high molecular weight proteins.

you can increase transfer by increasing time and/or current density (be careful not to exceed limits of the apparatus, if any (check the manual)), however, you may be sacrificing low molecular weight proteins (they'll blow through the membrane, this can be minimized by using smaller pore (0.2um or smaller) membranes).

another way to increase transfer is to add up to 0.05% sds to the transfer buffer (with methanol).

the methanol you added to the transfer buffer will actually reduce transfer efficiency by stripping sds from the proteins (this will enhance binding of the protein to the membrane), thereby reducing their mobility. adding sds will improve mobility.

if your films are darkening after they are processed then they are not being sufficiently fixed. either your fixer is depleted or you are fixing for too little time.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Mar 07 2017 3:38 pm

Screen Shot 2017-03-07 at 1.22.33 PM.jpg
I ran a WB again. The only differences from previous blots were O/N incubation at 4°C in TBS-T milk (blocking) and 1 h incubation at RT with primary antibody. Also, my samples were in sample buffer with DTT, frozen at -20°C. I took them out of -20°C and thawed it at RT, followed by a brief centrifugation before loading.

The first picture is with 10 min exposure time while the second is with 20 min. Please ignore the last well, it has a naive sample (non-treated sample). I see bands in lane 1-3 but they are big blobs, although the blobs are of the right size :P. In lane 4, I see a slight band while in lane 5, there is nothing. In lane 7-10 I see clear distinct band but they are of lighter intensity, this could be because they are from a different time point. But again in lane 9, I see a big blob.
Screen Shot 2017-03-07 at 1.22.38 PM.jpg


What can be done about the blobs? I loaded 15 µg of proteins, is it the reason for the blobs? What could have happened to lanes 11-13, did I load too little (15 µg) in them?
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Re: Western Blot - wrong size bands

Postby mdfenko » Mar 08 2017 3:06 pm

how do the bands look on a stained gel?

if they are diffuse then that's where the blobbiness (i made up a new word) comes from. if so then you can correct that with better stacking (do you use a stacking gel?) and/or by reducing the loading volume and/or reducing the loading height (do this by loading slowly from the bottom of the well rather than from the top, this way the sample won't mix with the electrode buffer).

if not, then you may be letting the gel sit for too long (including the time you equilibrate in transfer buffer before setting up the sandwich) before transfer. the band will diffuse.

or, the chemiluminscence may be too strong.
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