Hello there,
I'm doing protein concentration for WB assay using BioRad DC™ Protein Assay similar principle to Lowry assay. I have 8 samples and standard.I do them in triplicates. However, I've done the assay many times but I get the first column of all samples are higher (around double) than the other two columns. This happens only with samples but not standard.
Any idea?
Many thanks,
Abed
Hi,
I think the problem is pipetting error and your sample preparation.Whenever you want to determine protein concentration by gel,you have to be careful in sample preparation because some of you samples will stick to the wall of your tube or you pipetted excess into the first well than the other wells.I will suggest that since you intend to have 3 replicates,make sure you prepare 3.5x of your sample and not exact 3x so that you can load equal volume.Ensure consistent pipetting of your sample by using a same pipette range throughout and make use of a long tip for your loading into wells to avoid fusing of sample from the wells.
I hope this hints will help you.
Hi,
I think the problem is pipetting error and your sample preparation.Whenever you want to determine protein concentration by gel,you have to be careful in sample preparation because some of you samples will stick to the wall of your tube or you pipetted excess into the first well than the other wells.I will suggest that since you intend to have 3 replicates,make sure you prepare 3.5x of your sample and not exact 3x so that you can load equal volume.Ensure consistent pipetting of your sample by using a same pipette range throughout and make use of a long tip for your loading into wells to avoid fusing of sample from the wells.
I hope this hints will help you.
Hi, if I remember correctly the Lowry assay uses high pH. If high pH aggregates your proteins then you will get higher readings in the first wells.
Try the BCA Assay. It is a modification of the Lowry assay without the high pH.
@Kuo, unfortunately most of the researchers here do not have access to HPLC equipment that can be used for photometric analysis.
If you were in a well funded analytical lab, then yes you would have to use the best equipment to give the most accurate results.
But most scientists here on RG have modest means and require simple and inexpensive methods to get the answers they require for reproducible results.
Their methods will probably never be published in Analytical Biochemistry, but if they get reproducible results using simple methods then they get an A+ in my book.
Buffer pH leading to protein aggregation and pipetting errors could be one of the reason.
I agree with Dr. Stefansson. Try BCA microassay if you can't afford advanced techniques . It gives me really consistent and reliable reading.
If you are getting higher readings for sample alone it is very well because of the lysis buffer. Why don't you keep a buffer control for every column?
Another aspect to consider, in addition to all the previous answers, is the type of support you use for your quantification, if for example you use micro-ELISA or microwell systems, these plates must be new, not washed and then re -used, that generates an uncontrollable variability of variations between readings for the same sample. If you make Lowry in glass tubes and these have been washed with soap, the readings of your samples have no coherence. It seems simple or obvious, but necessary to save reagents and avoid repetitions of unnecessary protein quantification.
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