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how many times striping can b done of the blot


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9 replies to this topic

#1 pakbiochemist

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Posted 20 December 2011 - 11:17 AM

Dear friends,

I have not seen my tranfer by ponseau but on coommassie staing of the respective gel i obtained no bands does it mean that it is a sucessful transfer & secondly how many times i can strip my blot

Binsan

#2 bob1

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Posted 20 December 2011 - 11:47 AM

1) possibly - you could have "blown through" the proteins - that is the proteins may have migrated through the membrane during transfer, especially if you are trying to detect small proteins.
2) a few times, but it depends on the stripping procedure, the abundance of the proteins you are trying to detect, and the sensitivity of your antibodies.

#3 Biouday

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Posted 21 December 2011 - 03:37 AM

Dear friends,

I have not seen my tranfer by ponseau but on coommassie staing of the respective gel i obtained no bands does it mean that it is a sucessful transfer & secondly how many times i can strip my blot

Binsan


If you not seen any bands in ponseau then there is problem in protein transferring, Pls mention what is the size of the protein you are analyzing and if possible also mention about your transferring procedure.

Regarding stripping as Bob mentioned it depends upon procedure, am using commercial stripping buffer and once i used the same blot to detect 5 different proteins.If you planned to analyse many proteins in the same blot, then load more protein than usual.

#4 Biouday

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Posted 21 December 2011 - 03:39 AM

1) possibly - you could have "blown through" the proteins - that is the proteins may have migrated through the membrane during transfer, especially if you are trying to detect small proteins.
2) a few times, but it depends on the stripping procedure, the abundance of the proteins you are trying to detect, and the sensitivity of your antibodies.

Hi Bob, I like to know is it possible that small protein migrate through the membrane during transfer.

#5 pakbiochemist

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Posted 21 December 2011 - 11:30 AM

Dear Friends
i have blot 50 ug of histone protein and the transfer was overight at 30mAmp. I dddnt go for ponceau stain and directly add blocking sol followed by primary Ab.
My stripping solution is
100mM mercapto ethanol
10% SDS
1M Tris pH 6.7
water
How amny times can i strip y blot. and also advice me regarding the time for transfer of proteins from gel to membrane. & if anyone can share a paper regarding molecular weight and Ab concentration to b used and also the transfer rates of different proteins

Regards
Binsan

#6 bob1

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Posted 21 December 2011 - 12:01 PM

Hi Bob, I like to know is it possible that small protein migrate through the membrane during transfer.

Definitely. The pores in the membranes are much much larger (they are nearly the size of a bacterium) than any proteins you are likely to be detecting on a western. The liklihood of a protein migrating through depends on the membrane and transfer system you are using.

Dear Friends
i have blot 50 ug of histone protein and the transfer was overight at 30mAmp. I dddnt go for ponceau stain and directly add blocking sol followed by primary Ab.
My stripping solution is
100mM mercapto ethanol
10% SDS
1M Tris pH 6.7
water
How amny times can i strip y blot. and also advice me regarding the time for transfer of proteins from gel to membrane. & if anyone can share a paper regarding molecular weight and Ab concentration to b used and also the transfer rates of different proteins

Regards
Binsan

If you loaded only histone proteins, you won't see any other proteins, so why bother trying to detect them? If you actually loaded 50 ug of total cell lysate (as I suspect), then it may be worth stripping. Personally I disapprove of stripping, as you don't know how this affects the proteins that are on the membrane, and potentially alters any results you might have, unless you are very careful to keep the stripping conditions consistent.

You can't tell if the transfer has gone correctly, unless you ponceau stained the membrane. If you have consistently been able to transfer proteins using this method before, then you can be reasonably sure that you will have protein on the membrane. IIRC histones are likely to be able blow through the membrane as they are reasonably small (approx 15 kDa).

Your stripping will work, but you should determine the number of times you can do it for yourself. I would estimate that a maximum of 2 times (detect 3 proteins) would be about the limit!

#7 pakbiochemist

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Posted 22 December 2011 - 06:18 AM

Thank you friends for your help. today i hav blot my sample. i m keeping it in blocking solution overnight. does the incubation time in blocking solution alters the result or not.

Binsan

#8 mdfenko

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Posted 22 December 2011 - 06:50 AM

keeping it in block for longer periods may improve your background, it shouldn't hurt it.
talent does what it can
genius does what it must
i do what i get paid to do

#9 pakbiochemist

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Posted 22 December 2011 - 10:17 AM

hi frndz,
i did ponseau stain of my blot before blocking and the bands appear so cool. Thanks for the suggestion. i kept my histones for transfer at 75 v for 2 hrs.
Binsan

#10 mdfenko

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Posted 23 December 2011 - 12:04 PM

g-biosciences sells a solution that they claim will strip off antibodies but not harm the proteins bound to the membrane for multiple reprobing:

western reprobe plus

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talent does what it can
genius does what it must
i do what i get paid to do




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