i am working on bacterial amylase and trying to precipitate protein by 1:7 sample and ethanol method.when i precipitate the small quantity of protein in one tube(in one eppendorf i.e.125ul sample and 875ul of ethanol, precipitates dissolved in 200ul tris hcl buffer ) i get 70-75% recovery. but when i try to do the same with more quantity say 100ml of sample and 700ml of ethanol centrifuged in several tubes and precipitates pooled into 28ml of tris buffer the recovery of proteins is 50% please let me know what could be the reasons?
i tried using the sample with 50% recovery for gel chromatography where i used 500ul of sample in 10ml syringe eluted the sample with 30ml buffer and i got 15 fractions(8-22) of 0.5ml which showed activity for enzyme...on pooling up these fractions i got 7.5ml protein with 80% recovery...
should i proceed with ion exchange with the above protein and then do the characterization of enzyme?
Curious... that your protein yield is even lower than expected when using 1.25 X less tris buffer (28 instead of 35 mL).
In another technique (which could be used as an analogy to your situation) there is a reaction volume effect on RT reactions. The size of the reaction itself directly impacts the output. Meaning, that some processes are not scalable upwards (or downwards) as the physicality of the tube sizes used, coupled with the impact [e.g., of Brownian motion of the water and/or azeotrope of ethanol:water within the physical confines of the intended process] perhaps somehow influences the outcome of the process(es) undertaken.
E.g., this quote from a 2010 paper regarding RT reactions:
"...Reverse transcription efficiency varied up to 90-fold depending on the choice of reverse transcriptase, priming strategy, and assay volume..."
in: http://www.ncbi.nlm.nih.gov/pubmed/20226159
This leaves one to wonder what other processes could/can be affected likewise.
Curious that such a seemingly innocuous physical aspect of a process can indeed influence its outcome/efficacy. Tube geometry? When scaling up and down - perhaps be absolutely certain to use the 'Russian (matryoshka) doll approach' with the tubes you use. E.g. a 50 mL tube should have the exact congruence of geometry of a 15 mL tube, a 10 mL tube, a 5 mL tube, a 1.5 mL tube - etc. Sounds like voodoo but who knows. Not certain this holds in your situation - but, nonetheless food for thought?
It sounds like a classic "mass transfer" issue. You should look at your mixing methodology. The greater the volume, the greater the difficulty getting a homogeneous mixture. I'd try increasing the speed or length of time for your mechanical mixing step/s.
Hi
Do u have the same protein concentration in both cases? if not the same then try to extract your protein in a small volume say15- 20 mL each then combine the extracted protein and measure the protein concentration, let it stand longer time after the addition of ethanol. One last question what's the centrifuge speed u have used in both?
Hope this helps
@Abdulhadi Albaser
Hi i am keeping my sample in chilled ethanol for one hour in both the cases and the centrifugal speed is also the same for both.....12,000rpm for 1 hour
Hi
Add ethanol slowly with continuous gentle stirring (at 4C or on ice) have you done this step? use cold centrifuge, have you added a protease inhibitors to your sample? on top of that EtOH may denature your amylase so you will not see activity. Do you know the PI of your protein because if its closer to the pH of the used buffer then it will reduce the solubility of your protein.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue