3 replies to this topic

[porfirion]

can I measure protein concentration from RIPA lysate using NanoDrop reader at 280nm instead of standard methods such as BCA or Lowry, I compared BCA with this ones and results were quite different, let say 10 times higher in case of NanoDrop????

[Prep!]

well i dint quite understand the question completely but i can see that you are getting different conc by different methods. This is very normal. BCA is by a standard curve method and OD at 280 is by the absorbtion of aromatic amino acids. Moreover the cell lysate may have different proteins and the conc will vary ion both these nethods depending on the aromatic content. Also if you ar measuring at 280, what are you using as extinction value for calculating conc will also matter as these are a mix of proteins!!!
If i missed some important info in your question do write back.. or else i hope this helps.
In short for a mix of proteins, a colorimetric method is more reliable than the OD at 280. For the pure protein the values will be comparable.
Again it also depends on the standard that you are using. eg in bradford, the coomassie binds to basic amino acids and if you use bsa as a standard and your protein has less basic amino acids, it will be underestimated!!!
Best luck!!
Hope i dint confuse you!!

[klinmed]

porfirion, on Nov 17 2009, 12:56 PM, said:

can I measure protein concentration from RIPA lysate using NanoDrop reader at 280nm instead of standard methods such as BCA or Lowry, I compared BCA with this ones and results were quite different, let say 10 times higher in case of NanoDrop????

The NP-40 in RIPA has strong absorbance at A280. It is therefore not a good idea to measure protein concentration using this method.
Even if you can blank against buffer, this will result in low accuracy due to the reduced signal being available for the unknown sample. The BCA method is generally preferred for crude cell extracts since it tolerates detergents well.

Hope this helps

[Feelcontraire]

porfirion, on Nov 17 2009, 03:56 AM, said:

can I measure protein concentration from RIPA lysate using NanoDrop reader at 280nm instead of standard methods such as BCA or Lowry, I compared BCA with this ones and results were quite different, let say 10 times higher in case of NanoDrop????

RIPA has DOC, SDS, triton sometimes...and more stuff that interacts with the reactive- reaction/binding.

To avoid this components interference or any lysis buffer interference components you should go the dot-blot way.

Dilute BSA in your lysis buffer to a 20ug/ul concentration.

Simply prepare 2-factor 5-6 dilutions in water of your sample and control (BSA).

Dot on an Ethanol activated PVDF or nitrocelulose. Dry it to ensure equal adhesion. (Reactivate it in ethanol if it is PVDF). Was it briefly in water (this will remove the detergents). And insert it in Ponceau red (0,2%PonceauS, 3%TCA, 3%SAA). Leave it there 5min. Then place it in a water container to remove background, scan and measure densitometry with Image J.

This works with all buffers as the extra-stuff is removed after blotting, prior to detection.