BCA or Bradford? Which one is the better method to estimate the protein for western blot analysis?
These two are very commonly used protein estimation methods, But which one is the most efficient method to estimate the protein concentration for western blot?
Please give your valuable suggestions.
The bca tends to be more liner, but i have used the bradford to make my standard then print the curve on graph and find my unknown with great reproducable and accurcey. Silver stain can be the gold standard though.
I like the bradford, a little more time friendly depends on which kit you use to.
The key is to have the protein to be measured to be some where within the liner rang of the assay. If it is too concentrated, dilute it and retest, ... dont expect super accurate if to close to the end points of the assay. My typical range was like, 0.5, 1, 2, 3, 4, 5, 6 ,7 ,8 ,9 10 ug/ml of protein standars bsa curve if my lysate was too clise to .5 to 1., bad lysate, lost cells or protein somewhere., suspect sample. If like at 8 to 9 i would dilute 1 to 1 with buffer and re do protein assay.
Oh, spend time on your standard curve, pipette your best reproducable trip or quod samples and get avg and standard devations. You can get equ of line of best fit and use equ to find concentration of your unknown. But i like to make a graph and find my concentration on it from abs. Vs con.
Best wishes, good luck
Charle
I agree that BCA works well, but just from a point of conveninece it is possible to do BCA in a 96 well plate where as mostly Bradford needs to be done using cuvettes due to the volumes needed.
I have used both bradford and BCA, I think both work well. As Harald mentioned, BCA is less sensitive to detergents. However, if you dilute your samples, then RIPA does not influence bradford. I did bradford in 96w format, you use 200ul of bradford and add 2ul of standard or sample (10x diluted in PBS). In case of BCA, the recommended volumes of sample is 10-25ul, which is quite more than required for bradford. So I also dilute samples 10 times and use 25ul for 200ul of BCA. I always do the measurements in duplicates. Also bradford is fast because there is no incubation time whereas for BCA its 30 mins. So there are advantages and disadvantages...but both work equally well. Good luck!
Detergents bind the Coomassie dye and therefore artificially contribute to signal.Reducing agents react with the Cu2+ ion in the BCA mixture and therefore artificially contribute to signal.
Therefore:
Use the BCA assay if your samples contain non-ionic detergents (Triton, NP-40, Tween, etc.). But use the Bradford assay if your samples contain reducing agents (DTT, BME, TCEP, etc.).
If your samples contain both detergents and reducing agents? Try the Pierce 660nm reagent:
http://www.thermofisher.com/order/catalog/product/22660?gclid=CjwKCAjwk4vMBRAgEiwA4ftLs8LKMPfGP6CRDyfp4vvnNAhV36J6Lo1yxmoWqMQF_5TYkFyheM6MvRoCsHYQAvD_BwE&s_kwcid=AL!3652!3!85589465054!b!!g!!&ef_id=V6zavQAABDTnZz8m:20170803145012:s
Thank you so much for the suggestions. Currently, I am using Bradford reagent for protein estimation. I will also estimate same protein sample with BCA kit from my neighbour lab to check the difference between two methods.
Again thanks everyone for your suggestions!!
Pounami, I like your idea too. Instead of using 1ml of Bradford we can scale down the volume to 200ul. Thanks.
Both formats are fine as long you scale down the dilutions and use the dilution factors in the final analysis. Thanks.
I would use Bradford as it is a bit easier to use, especially if you are using reducing agents. BCA is less sensitive to your detergents so its a bit more robust if you're using non-ionic agents (as stated before).
I would use Bradford as it is a bit easier to use, especially if you are using reducing agents. BCA is less sensitive to your detergents so its a bit more robust if you're using non-ionic agents (as stated before).
I would use Bradford as it is a bit easier to use, especially if you are using reducing agents. BCA is less sensitive to your detergents so its a bit more robust if you're using non-ionic agents (as stated before).
Try the biuret method given in Stephen Zamenhof: Preparation and assay of deoxyribonucleic acid from animal tissue Methods in Enzymology. 3: 696-704. DOI: 10.1016/S0076-6879(57)03446-1
This is a quite specific method, as it depends on presence of -NH-CO- bond in the sample. A drawback is the high concentration of hydroxide (needs careful handling). However, it is quite sensitive, specific and inexpensive.
We routinely use BCA for protein estimation in our lab, followed by WB.
Hello Utsav,
For quantifying membrane proteins, we would normally use BCA because we require detergents to solubilise our membrane proteins. BCA is less sensitive to detergents than Bradford. Bradford is fine for quantification of other types of proteins (particularly soluble proteins) and those that require reducing agents. It all depends on what type of protein you would like to quantify. I hope this answer helps. All the best !
Joan
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