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how can i explain the different pattern from my labmate's one

western blot WB pattern

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#1 Miaaa

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Posted 10 August 2014 - 08:27 PM

I did WB with my labmate under same conditions(each step, sample, same antibody..etc)

but the result is perfectly different from my labmate’s one.

membrane’s pattern is different..

 

also, I checked my membrane after transfer. it has no problem.

so I think problems which can cause trouble in it is from after the blocking

 

 

 

I cant figure out what is the exact problem. Could you please suggest possible reasons and how to overcome to these problems?

 

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#2 bob1

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Posted 11 August 2014 - 12:14 AM

I see membranes that would probably look the same if they had the exposures altered a bit (longer for the one on the right)...



#3 Miaaa

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Posted 11 August 2014 - 12:53 AM

I see membranes that would probably look the same if they had the exposures altered a bit (longer for the one on the right)...

 

@bob1

 

i did exposure longer. but...not like the one on the left..TT...just looks like tubulin without any increasement or reduction..



#4 bob1

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Posted 11 August 2014 - 03:35 AM

So... tell us what you did (details - sample types, gel%, blotting systems, loading buffer, boiled?, membrane type, antibodies, block, times, washes, detection chemistry etc.) and what your labmate did - it's very hard to trouble shoot unless we know what you are doing.



#5 Miaaa

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Posted 11 August 2014 - 06:09 PM

So... tell us what you did (details - sample types, gel%, blotting systems, loading buffer, boiled?, membrane type, antibodies, block, times, washes, detection chemistry etc.) and what your labmate did - it's very hard to trouble shoot unless we know what you are doing.

 

@bob1 thanks for your kind.

 

our sample is lung tissue lysate from mice.

we made sample via BCA assay and boiled it for 5min at 99º before we ran.

we both ran 30ug lysate in 8% gel. 70V→100V→120V

next, we trasferred to NC membrane. after it is finished, I checked my membrane via Ponceau staining. it had no any problem.

 

blocking with 5% skim milk for 1hour

washing with TBST for 10min (three times).

 

primary ab O/N (1:1000)

washing with TBST for 10min(three times).

secondary ab for 1hour (1:5000) (mouse)

washing as I did before

ECL.

 

---------------------------------------------------------------

but the result is totally different.

so I tried many different methods to overcome this trouble.

(changing antibody titer, transfer time..etc)

 

but I don't know the reason why this problem happened...

 

only one which caused this problem is my hand........so weird...how do I overcome it?......my labmate also doesn't know this reason.


Edited by Miaaa, 11 August 2014 - 06:22 PM.


#6 bob1

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Posted 12 August 2014 - 12:01 AM

Assuming that the samples were taken from the same aliquots, it is possible that you have some handling differences somewhere - perhaps one of you is keeping the protein on ice, and the other not, or that one is taking sample that isn't mixed properly after thawing.

 

Often differences in staining are due to miscalculated dilutions of antibodies - too much secondary can give you multiple non-specific bands.  Try repeating it using dilutions made by one person only and see how you go.

 

Sometimes, especially for wet transfers, if one of the membranes is closer to the black side (cathode) in a tank with more than one membrane (e.g. biorad protean transfer tanks, it will transfer better than the membrane which is closer to the anode (red). 

 

Ponceau staining can inhibit antibody binding, you need to wash it off fully before probing.



#7 mdfenko

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Posted 12 August 2014 - 04:05 AM

also, in what medium do you prepare your antibodies?

 

if you include blocking agent in your antibody solutions (always a good practice) then you probably don't have to wash between the block and primary antibody and you may reduce nonspecific binding of the primary antibody.


Edited by mdfenko, 12 August 2014 - 04:06 AM.

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#8 Miaaa

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Posted 12 August 2014 - 09:49 PM

also, in what medium do you prepare your antibodies?

 

if you include blocking agent in your antibody solutions (always a good practice) then you probably don't have to wash between the block and primary antibody and you may reduce nonspecific binding of the primary antibody.

thanks for your good advice!

@mdfenko

 

my antibody was in TBST not mixed with BSA or Skim milk.

I will try as what you said next time...but is it effective?

I heard from somewhere that the method can inhibit antibody-antigen binding.



#9 mdfenko

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Posted 13 August 2014 - 03:58 AM

we've always included blocking agent with our antibodies.

 

if you get reduced result that would be due to the block pre-absorbing species of the antibody that bind to blocking agent, decreasing background and other non-specific binding.


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