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Coomassie gel after semi-dry transfer - normal like this?


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#1 Podge

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Posted 22 August 2013 - 08:39 AM

Hey guys,

 

started using Western Blots last week and have a question regarding my transfer.

 

This is what the gel looks before and after transfer to a Nitrocellulose membrane using a semi-dry transfer using "NuPage 2x transfer buffer" with 10% Methanol in it (according to the manufacturers protocol for semi-dry blots). 5min @10V, 45min @20V

 

Please see the attached coomassie gels. On the one post transfer I have these funny looking bubbles and I am wondering if that is something to do with the transfer.

Also I have noticed there is still quite some protein left on the gel.

 

I appreciate your help.

 

Thanks

Attached Thumbnails

  • E12+HSD++ctrl Joe pre-transfer 200813.jpg
  • E12+HSD++ctrl Joe post-transfer 200813.jpg


#2 mdfenko

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Posted 22 August 2013 - 09:33 PM

are these identical or the same gel?

 

it is normal to see incomplete transfer, especially of high molecular weight proteins.

 

how does the membrane look? (ponceau s staining can let you see how well the transfer went, as well as the prestained standards)

 

some of what you are seeing may be normal after transfer (try staining other gels after transfer), but it looks like you may have had some bubbles during transfer (the membrane can tell you if they affected the transfer).

 

you may also be seeing some uneven transfer from side-to-side. if this is the case, it can be caused by uneven contact, uneven pressure, dirty electrode(s).


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#3 Podge

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Posted 23 August 2013 - 02:11 AM

are these identical or the same gel?

 

it is normal to see incomplete transfer, especially of high molecular weight proteins.

 

how does the membrane look? (ponceau s staining can let you see how well the transfer went, as well as the prestained standards)

 

some of what you are seeing may be normal after transfer (try staining other gels after transfer), but it looks like you may have had some bubbles during transfer (the membrane can tell you if they affected the transfer).

 

you may also be seeing some uneven transfer from side-to-side. if this is the case, it can be caused by uneven contact, uneven pressure, dirty electrode(s).

 

These are two identical gels. I stained one and then used one for the transfer and Western labelling.

The membrane looked good to me. The one sample in the middle was nice and good to see, the other samples were very faint. But it didn't seem like there were big areas not stained affected by air bubbles. All of the prestained standard had transfered over to the membrane and in the very small parts even a little bit over to the filter.

 

Is there a way to improve the transfer? Longer and less voltage?

Also: I just put the nitrocellulose membrane in the buffer for about a minute and then stacked the sandwich together. Is that ok?

 

Thank you so much for your help.



#4 mdfenko

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Posted 23 August 2013 - 10:14 AM

then the bubbles probably formed during removal.

 

until you confirm that no protein passed through the membrane you should use a backing membrane. what pore size do you use? i've had good results with 0.2um for general purposes (most recommend 0.45um).

 

with our semi-dry unit, a specific current density (A/cm2 is recommended. you should follow the recommendations for your specific unit (rtm). there is usually a good troubleshooting section. without knowing which apparatus you use it would be difficult to make anything more than a general comment.

 

you only need to soak the nc membrane in buffer long enough to fill the pores and wet it. i float the membrane on the buffer (without bubbles) until the pores fill (buffer pools on the surface) then briefly submerge and transfer onto the sandwich.

 

i've also seen stain from the standard on the filter. i think it comes from stain coming off the standard rather than protein passing through the membrane (at least, with 0.2um pore membrane), but i could be wrong.


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#5 Podge

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Posted 27 August 2013 - 06:01 AM



then the bubbles probably formed during removal.

 

until you confirm that no protein passed through the membrane you should use a backing membrane. what pore size do you use? i've had good results with 0.2um for general purposes (most recommend 0.45um).

 

with our semi-dry unit, a specific current density (A/cm2 is recommended. you should follow the recommendations for your specific unit (rtm). there is usually a good troubleshooting section. without knowing which apparatus you use it would be difficult to make anything more than a general comment.

 

you only need to soak the nc membrane in buffer long enough to fill the pores and wet it. i float the membrane on the buffer (without bubbles) until the pores fill (buffer pools on the surface) then briefly submerge and transfer onto the sandwich.

 

i've also seen stain from the standard on the filter. i think it comes from stain coming off the standard rather than protein passing through the membrane (at least, with 0.2um pore membrane), but i could be wrong.

 

Hey. thanks for your answer.

I use a 0.2 micron Nitrocellulose membrane. Also used two together in one run to see if it passes through and only in the low molecular bottom part a bit of the protein was transferred to the 2nd membrane.

I use a Bio-Rad transblot SD semi-dry cell. Pretty standard in most labs. Checked the manual and I don't think I go over the max. allowed A/cm2.

 

Here is a Ponceau Pic of the membrane just after transferring. There is faint bands on the other lanes, but hard to see in that pic: http://s1.directuplo...27/uc4ol8q4.jpg



#6 mdfenko

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Posted 28 August 2013 - 04:10 PM

you may be able to improve transfer by using a gradient gel to separate the proteins. it should improve the transfer efficiency of the high molecular weight proteins and (slightly) reduce the transfer efficiency of the low molecular weight proteins (we routinely use gradient gels).

 

you can also include up to 0.05% sds in the transfer buffer to aid transfer of high molecular weight proteins (you may want to increase methanol to 20%, as well).


Edited by mdfenko, 28 August 2013 - 04:11 PM.

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#7 Podge

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Posted 29 August 2013 - 05:09 AM

Hey,

 

I already use gradient gels, so that shouldn't be an issue. I will add some SDS and bump up the Methanol to 20% and see how that works. Will get back to you when I have more results on that.

 

One other question:

just from your experience which of the lanes in the commassie gel would you think is 20ug of protein. the big fat one in the middle or the other ones? I somehow doubt my Bradford results.... another issue I have...

 

Thanks!


Edited by Podge, 29 August 2013 - 05:55 AM.


#8 mdfenko

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Posted 29 August 2013 - 04:04 PM

that's hard for me to say. we use much larger gels where we use at least 50ug of total protein (crude) and don't get staining as strong as your strongly stained lane.


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