Western Blot - wrong size bands

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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 11:22 am

there is always protein left on the gel (try silver staining after you don't see anything with coomassie).

the type of transfer apparatus may limit the time you can use to transfer the proteins. we had a semi-dry apparatus which was not to be used for more than 60 minutes and current density of 2.5mA/cm2. but that was sufficient to transfer pretty much any protein in which we were interested.

we used 0.2um pore membranes to ensure that smaller proteins didn't just blow through the membrane.

we used gels that were on the large side and routinely loaded 50-100ug of crude extracts.

if you want to load less then you will have to at least partially purify the protein.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 12:55 pm

mdfenko wrote:there is always protein left on the gel (try silver staining after you don't see anything with coomassie).

the type of transfer apparatus may limit the time you can use to transfer the proteins. we had a semi-dry apparatus which was not to be used for more than 60 minutes and current density of 2.5mA/cm2. but that was sufficient to transfer pretty much any protein in which we were interested.

we used 0.2um pore membranes to ensure that smaller proteins didn't just blow through the membrane.

we used gels that were on the large side and routinely loaded 50-100ug of crude extracts.

if you want to load less then you will have to at least partially purify the protein.


How do I purify the protein? Also, is there any disadvantage of loading large amount?
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 1:02 pm

I have two more questions in mind, and I don't want to open a new topic, so I'm asking it here.

I'm trying to use a different antibody on the same membrane. The first antibody that I used was produced in mice and the new antibody that I want to use is produced in rabbit. Do I need to strip the membrane? When is stripping of the membrane necessary?

After using the primary antibody once in TBS-T milk, I collect the antibody and add NaN3 and store it. Should it be stored at -20°c or 4°C is fine? How many times can I use the same antibody?

Thanks :)
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 2:50 pm

newbie17425 wrote:How do I purify the protein? Also, is there any disadvantage of loading large amount?

I'm trying to use a different antibody on the same membrane. The first antibody that I used was produced in mice and the new antibody that I want to use is produced in rabbit. Do I need to strip the membrane? When is stripping of the membrane necessary?

After using the primary antibody once in TBS-T milk, I collect the antibody and add NaN3 and store it. Should it be stored at -20°c or 4°C is fine? How many times can I use the same antibody?

there are a number of ways to partially purify the protein. some common ways are to fractionally precipitate the protein of interest with ammonium sulfate or acetone or polyethylene glycol. you can also pass the extract through gel filtration (separation by size), ion exchange or chromatofocusing (by charge), hydrophobic interaction or hilic (hydrophobicity or hydrophilicity).

the disadvantages of loading a large amount are the introduction of too much lipid (in crude extracts, especially from brain), salts, buffers and even nucleic acids. these can all disrupt the normal electrophoretic pattern (come to think of it, the apparent depression of the size of your protein may be caused by one of these).

you can reuse the same blot if you kill the hrp from the first secondary antibody (sodium azide will do the trick). the new secondary will be against a different species so it won't bind to the first primary. stripping is necessary if the primaries are from the same species.

storage temperature depends on the length of time for which you want to store. the milk will sour at 4C in a few days (the antibody will also denature). the number of times you can use it depend upon how fast you deplete it. you will experience some variation in results from run to run. personally, i decided that i use so little at one time that i just prepare it fresh for each blot.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 17 2017 3:00 pm

I appreciate your patience in answering my questions. Thank you :D :D

mdfenko wrote:the disadvantages of loading a large amount are the introduction of too much lipid (in crude extracts, especially from brain), salts, buffers and even nucleic acids. these can all disrupt the normal electrophoretic pattern (come to think of it, the apparent depression of the size of your protein may be caused by one of these).

I'm lost again, do you think I should load less amount then?

mdfenko wrote:you can reuse the same blot if you kill the hrp from the first secondary antibody (sodium azide will do the trick). the new secondary will be against a different species so it won't bind to the first primary. stripping is necessary if the primaries are from the same species.

Ok. But for example, my TLR7 was produced in mouse after developing, I washed it and added ACTß, also produced in mouse. Do I need to strip the membrane here?

mdfenko wrote: storage temperature depends on the length of time for which you want to store. the milk will sour at 4C in a few days (the antibody will also denature). the number of times you can use it depend upon how fast you deplete it. you will experience some variation in results from run to run. personally, i decided that i use so little at one time that i just prepare it fresh for each blot.
[/quote][/quote]

Should I store them at -20°C then? I don't have much antibody left and we are financially low to buy more antibodies.

Thanks once again !!
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 17 2017 3:16 pm

do you think I should load less amount then?
i would try more and see what happens (you could run a concentration series to find the optimal amount to load).
my TLR7 was produced in mouse after developing, I washed it and added ACTß, also produced in mouse. Do I need to strip the membrane here?
the secondary was produced against mouse igg, it shouldn't react to anything else.
Should I store them at -20°C then? I don't have much antibody left and we are financially low to buy more antibodies.
again, it depends on how long you plan to store it and how many times you'll use it. while storage at 4C is only good for the short term, repeated freeze/thaw will also deplete the antibody.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 21 2017 7:14 am

mdfenko wrote:
my TLR7 was produced in mouse after developing, I washed it and added ACTß, also produced in mouse. Do I need to strip the membrane here?
the secondary was produced against mouse igg, it shouldn't react to anything else.

sorry, misread this question...
yes, you must strip before adding the antibody to actbeta if it was made in the same species as the antibody to tlr7.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 21 2017 7:29 am

mdfenko wrote:
mdfenko wrote:
my TLR7 was produced in mouse after developing, I washed it and added ACTß, also produced in mouse. Do I need to strip the membrane here?
the secondary was produced against mouse igg, it shouldn't react to anything else.

sorry, misread this question...
yes, you must strip before adding the antibody to actbeta if it was made in the same species as the antibody to tlr7.


Thanks for the clarification :)
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 23 2017 8:54 am

The problem with the wrong band size is solved now. I used a different antibody from Novus and it worked properly. However, 3 out of my 7 samples showed band and I loaded 15 µg of proteins. Should I load more i.e. 20-25 µg of proteins instead of 15 µg? Also, there was some background, how can I clear the background. I block using 5% milk in TBS-T and wash it with TBS-T and TBS after incubation in secondary antibody.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 23 2017 10:59 am

if the samples that showed bands was weak or if there may be traces of the band in the others then increasing sample load should improve the results.

you might improve the background by increasing blocking time or concentration. i don't add tween to my blocker. tween supposedly reduces non-specific binding. when blocking, i want as much binding as possible without disrupting the transferred proteins.

i also add normal serum from the species in which the secondary antibody is produced to the block and antibody solutions.

another thing, are you using a biotinylated system for detection? if so then you should change your block. milk contains biotin and will increase your background (milk should not be used in biotin-(strept)avidin systems and phosphoprotein detection systems (casein is highly phosphorylated)).
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 23 2017 11:16 am

mdfenko wrote:if the samples that showed bands was weak or if there may be traces of the band in the others then increasing sample load should improve the results.


I'm attaching an image of the blot here. There are three bands, but cannot be seen properly here as there is high background. From L to R, 1, 2, and 4 has bands and the band in the 4th lane is the sharpest.

mdfenko wrote: you might improve the background by increasing blocking time or concentration. i don't add tween to my blocker. tween supposedly reduces non-specific binding. when blocking, i want as much binding as possible without disrupting the transferred proteins.


Currently, I'm blocking for 1h at RT. How much should I increase the time to, 2 h?

i also add normal serum from the species in which the secondary antibody is produced to the block and antibody solutions.

mdfenko wrote:another thing, are you using a biotinylated system for detection? if so then you should change your block. milk contains biotin and will increase your background (milk should not be used in biotin-(strept)avidin systems and phosphoprotein detection systems (casein is highly phosphorylated)).

No, I don't use a biotinylated system. Should I try using BSA in TBS-T instead of milk for blocking?
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 23 2017 2:42 pm

i use a mixture of bsa and normal serum for my block and normal serum in the antibody solutions. blocking is for an hour at room temperature or overnight at 4C with gentle agitation.

your background is blotchy, rather than smooth and even. that normally indicates that there is insufficient agitation to allow the blocking and antibody solutions to circulate evenly. the membrane may be sticking to the vessel.

also, the vessel in which you incubate the blots may be dirty.

background like that can also be caused by uneven contact in the transfer sandwich.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 23 2017 3:23 pm

What can be done to correct this blotchy background?

However, when I used Actin, it was without any background. Both the membranes were treated the same way.
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Re: Western Blot - wrong size bands

Postby mdfenko » Feb 24 2017 7:28 am

part of the difference in background (probably the major part) between your protein of interest and actin is the specificity of the primary antibody.

while we're trying to figure this out, you may want to download some free handbooks and guides which will help with troubleshooting:

boster bio

ge lifesciences (under "protein analysis")

emd-millipore protein blotting handbook

was the result with tlr7 a one-time result or have you duplicated it? if it's one time then i would suspect that the background is due to the membrane sticking to the vessel.
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Re: Western Blot - wrong size bands

Postby newbie17425 » Feb 24 2017 7:36 am

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?
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