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Bradford and triton lysis buffer


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#1 itaimo

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Posted 04 July 2011 - 01:59 AM

I've read that Bradford protein quantification, is not working well with sample that extract using a lysis buffer with Triton, Or NP-40, because that the Triton and NP-40 react with the Bradford reagents and produce blue color.
http://www.protocol-...osts/13090.html

since I'm using it in order to dilute the protein samples, so that I will load equal amount of proteins to each well,
is it still possible to use Bradford, because all the samples are with the same concentration of Triton, Or NP-40, and the error will be the same ?

Is it possible to add Triton to the BSA protein samples, so that the protein level calculation will be Accurate ?

Edited by itaimo, 04 July 2011 - 02:57 AM.


#2 GNANA

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Posted 04 July 2011 - 02:33 AM

what percent of Triton or Np40 you are using??? because i am using the same and had no prob with bradford compatibility, i think you shdnt have any prob untill atleast 1% of those.


I've read that Bradford protein quantification, is not working well with samples that extraction using a lysis buffer with Triton, Or NP-40, because that the Triton and NP-40 react with the Bradford reagents and produce blue color.
http://www.protocol-...osts/13090.html

since I'm using it in order to dilute the protein samples, so that I will load equal amount of proteins to each well,
is it still possible to use Bradford, because all the samples are with the same concentration of Triton, Or NP-40, and the error will be the same ?

Is it possible to add Triton to the BSA protein samples, so that the protein level calculation will be Accurate ?


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#3 itaimo

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Posted 04 July 2011 - 03:06 AM

1% Triton, dilute from Triton X100 solution.

The Lysis buffer for animal tissues is:
10mM Tris
50mM NaCl
1mM EDTA
1mM EGTA
1% Triton X100
0.1% BSA

I'm using an hemoginizer, Is it better to use after that also a sonicator ?
Does it crucial to use sonicator in order to extract nuclear proteins ?

what percent of Triton or Np40 you are using??? because i am using the same and had no prob with bradford compatibility, i think you shdnt have any prob untill atleast 1% of those.



I've read that Bradford protein quantification, is not working well with samples that extraction using a lysis buffer with Triton, Or NP-40, because that the Triton and NP-40 react with the Bradford reagents and produce blue color.
http://www.protocol-...osts/13090.html

since I'm using it in order to dilute the protein samples, so that I will load equal amount of proteins to each well,
is it still possible to use Bradford, because all the samples are with the same concentration of Triton, Or NP-40, and the error will be the same ?

Is it possible to add Triton to the BSA protein samples, so that the protein level calculation will be Accurate ?



#4 GNANA

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Posted 04 July 2011 - 05:57 AM

I think your recipe seems to be bradford friendly, but to extract nuclear proteins i dont know whether it is enough efficient, i guess you need to increase the salt conc more. may be others here who has done nuclear protein extraction can give you more suggestions to improve the efficiency of your recipe..then regarding homogenising i think its upto you, either sonication or even syringing does the same.
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#5 mdfenko

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Posted 15 July 2011 - 05:56 AM

i would be more concerned with the effect of the bsa in the buffer on any protein assay.
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#6 itaimo

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Posted 11 September 2011 - 12:08 AM

What is the purpose of the BSA in the lysis buffer ?
Other lysis buffers that i've found non't contain BSA. The BSA creates a thick line at about 50KDa in bradford test (BCA kit), a line that is not exist in other lysis buffers.

#7 bob1

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Posted 11 September 2011 - 06:14 PM

BSA is probably helping buffer the sample a bit - but it will definitely react with the bradford reagents and give you a false measure of the amount of protein you have put in.

#8 allynspear

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Posted 21 September 2011 - 01:03 PM

The BSA is probably not doing anything substantial in the lysis buffer and it probably is affecting the Bradford. The bigger issue here is that 1% triton is not sufficienct to lyse most nuclei, so if you are looking for nuclear proteins you will need a more agressive lysis buffer. I would recommend looking for a RIPA buffer that contains 0.5% SDS, Sodium Deoxycholate, and 1% Triton X-100. These type of lysis buffers will release protein from all subcellular compartments and most membranes. However, these lysis buffers will affect Bradford reactions, but all you need to do is perform your bradford on a 1/10 dilution of sample in water. This should keep your protein concentration in the linear range of a Bradford but dilute out the buffer components below the interfering concentrations.

Best of Luck.

#9 itaimo

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Posted 21 September 2011 - 08:03 PM

I think that the BSA purpose might be to reduce the sample protein lose because of protein binding to the tubes in small protein concentrations.
If you add the sample protein to BSA, than the BSA will bind also to the tube and less protein sample will be lost.

For nuclei lysis, the addition of a sonication process should do the job.

BCA protein detection kit (BCA Protein assay - Thermo #23227) is supposed not to be effected by Triton, Or NP-40.

Edited by itaimo, 21 September 2011 - 08:07 PM.


#10 almost a doctor

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Posted 26 September 2011 - 08:15 AM

BCA protein detection kit will not be affected by the detergents however it will still be affected by the BSA in your lysis buffer.
I agree that the BSA is probably there to help prevent protein loss, but as long as you have 0.1% BSA in your sample, you are not going to be able to measure the concentration of your protein samples.

0.1% BSA = 1mg/ml of BSA which is going to mask any other protein you have there, specially if their concentration is low.

#11 JeremyDNA

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Posted 12 November 2013 - 09:39 PM

I'm a little late to the party but will offer help to others planning similar assays.

Coomassie blue (Bradford reagent) reacts with Triton x-100 and other detergents, so will cause significant interference. In many cases, the interference is high enough to destroy any hope of building a reliable standard curve, which is critical for protein quantitation.

To check at which concentration of Triton x-100 in a lysis buffer is suitable for a Bradford assay, I ran a serial dilution of:

2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.01562%, and 0% Triton x-100 in HBSS.

I found that loss of interference occurred near 0.0625%, which could explain why many lysis buffer recipes call for 0.05% Triton x-100 - I gather such buffers were designed to be Bradford compatible. In summary, I wouldn't exceed 0.05% if you want /need to use a Bradford assay.




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