9 replies to this topic

[itaimo]

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.

[GNANA]

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.

itaimo, on 04 July 2011 - 01:59 AM, said:

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 ?

[itaimo]

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 ?

GNANA, on 04 July 2011 - 02:33 AM, said:

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.

itaimo, on 04 July 2011 - 01:59 AM, said:

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 ?

[GNANA]

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.

[mdfenko]

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

[itaimo]

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.

[bob1]

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.

[allynspear]

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.

[itaimo]

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.

[almost a doctor]

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.