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Problem with SDS-PAGE gel

sds-page Gel polyacrylamide protein

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#1 bio_film

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Posted 11 August 2013 - 08:44 AM

Dear all, 

              I am having problem with running proteins samples on SDS-PAGE. I don't know the cause but I can show you the problem. I am uploading the pictures of the problem. I read earlier about how to troubleshoot SDS-PAGE. I have tried new Sample buffer and lower the APS and TEMED conc. for stacking gel I have also tried not to screw the plates tightly but nothing worked. First picture is just at the start of the SDS-PAGE and second one of the same PAGE after a while. 

I would highly appreciate if someone could explain the problem to me. Its really messing up my experiments.

 

Thank you 

 

Untitled.gif Untitled2.gif



#2 Adrian K

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Posted 11 August 2013 - 09:05 AM

What is the voltage you run your SDS-PAGE? Do you use cooling water circulator?


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#3 bio_film

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Posted 11 August 2013 - 10:49 AM

What is the voltage you run your SDS-PAGE? Do you use cooling water circulator?

I run at 120V(constant). I never used cooling water circulator in fact we never do in the lab except during transfers. I am having this problem for last 3 PAGEs I have done, not before. Thanks



#4 origami

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Posted 11 August 2013 - 11:38 AM

hi,

 

i had sth similar recently and then i realized that instead for a stock buffer 1.5M Tris pH 8.8 i was using 1M Tris pH8.8.

 

just be sure for your stock solutions, your running buffer to be fresh, check the mA instead the Voltage, boil your samples @95C for 5-10min and spin them before loading onto the gel.



#5 bio_film

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Posted 11 August 2013 - 11:58 AM

hi,

 

i had sth similar recently and then i realized that instead for a stock buffer 1.5M Tris pH 8.8 i was using 1M Tris pH8.8.

 

just be sure for your stock solutions, your running buffer to be fresh, check the mA instead the Voltage, boil your samples @95C for 5-10min and spin them before loading onto the gel.

We only have 1.5M Tris pH8.8 so thats perhaps is not the problem. We prepare our running buffere fresh. I will check again for these things. Did change to 1.5M solved the problem? did you change anything else?

 

thanks



#6 Shefali Desai

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Posted 12 August 2013 - 01:20 AM

Do you degas your gel solu. before pouring? You should do that as air bubbles do create problems. Looks like your gel has not set properly. Was there a minute leak?



#7 bio_film

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Posted 12 August 2013 - 05:32 AM

Do you degas your gel solu. before pouring? You should do that as air bubbles do create problems. Looks like your gel has not set properly. Was there a minute leak?

I don't degas, In fact I never had to. This problem is recent. I believe it was set properly cause the remaining gel in falcon was hard as a rock. There is always a bit of leakage which is normal. I still dont know what is the problem.



#8 bio_film

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Posted 12 August 2013 - 09:16 AM

Is there someone who could look at the pictures and tell me what could be wrong?



#9 origami

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Posted 12 August 2013 - 12:37 PM

 

hi,

 

i had sth similar recently and then i realized that instead for a stock buffer 1.5M Tris pH 8.8 i was using 1M Tris pH8.8.

 

just be sure for your stock solutions, your running buffer to be fresh, check the mA instead the Voltage, boil your samples @95C for 5-10min and spin them before loading onto the gel.

We only have 1.5M Tris pH8.8 so thats perhaps is not the problem. We prepare our running buffere fresh. I will check again for these things. Did change to 1.5M solved the problem? did you change anything else?

 

thanks

 

yeap it did change...that period i was doing co sedimentation assays and i had to be really careful with my gels because i wanted to quantify the bands...which means that i had to have perfect made gels....and i dodnt know why i didnt pay attention the concentration of the stock buffer...i took it as a granded until one colleugue noticed this.



#10 mdfenko

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Posted 13 August 2013 - 03:50 PM

are these the same samples as you've run in the past?

 

if not, is there salt with the sample?

 

are any of the solutions you are using newly prepared (just before your gels went south)?

 

how old is the sds?


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#11 bio_film

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Posted 20 August 2013 - 03:21 PM

are these the same samples as you've run in the past?

 

if not, is there salt with the sample?

 

are any of the solutions you are using newly prepared (just before your gels went south)?

 

how old is the sds?

Thanks for reply. 

the sample are treated the same way as in the past but the antigen is different. so basically samples are not different.

i dont understand what is meant by salts

We dilute from stock running and transfer buffer

SDS is new.



#12 mdfenko

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Posted 21 August 2013 - 02:23 PM

in what medium is your antigen prior to adding sds sample buffer?

 

salts such as nacl, kcl, na-acetate, k-acetate, etc.

 

how old are the stocks.

 

new lot of sds or newly prepared solution from off-the-shelf lot?


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#13 bio_film

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Posted 27 August 2013 - 07:28 AM

in what medium is your antigen prior to adding sds sample buffer?

 

salts such as nacl, kcl, na-acetate, k-acetate, etc.

 

how old are the stocks.

 

new lot of sds or newly prepared solution from off-the-shelf lot?

I used RIPA lysis buffer and proteins are in it.

yes we use NaCl in RIPA

stocks are fresh 

yes i used newly prepared sds.



#14 mdfenko

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Posted 28 August 2013 - 04:00 PM

newly prepared from off the shelf lot of sds? your sds may be decomposing.

 

also detergents in the ripa can affect sds binding, salts in ripa can affect migration and heating of the gel.

 

you may want to confirm that the new stocks are prepared correctly (not intended as an insult, sometimes stuff happens).


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