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Purpose: To understand and gain hands-on experience using centrifugation and ammonium sulfate to precipitate protein. To gain additional experience working with proteins and manipulating them safely using the rules for working with proteins. Also to gain additional experience/practice with solution preparation by making 50mM Geoes pH 7.5 at 4 degrees C, 0.33M Sucrose.



Materials:  

Gloves

250mL graduated cylinder

200mL Beaker

Sucrose (Fisher 11-2-2000, Lot 006196 FW: 342.30g/mol)

Hepes (Sigma 01/12/2001, Lot 10K5423 FW: 283.3g/mol)

diH2O

1M NaOH

Weigh boat

Analytical balance (A&D 3556692)

pH Meter (Accumet #7)

Stir bar

Stir plate

Transfer pipette





Procedure: 

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First, we do the math:

 

We're making 200mLs of 50mM Hepes (pH 7.5), 0.33M Sucrose.



Dimensional analysis (conversions - Thank you Mike Naffzigger, PCC Chem Instructor): 

(50mM/1L)(1M/1000mM)(Hepes 283.3g/Mol)(1L/1000mL)(200mL/1) = 2.383g of HEPES

(0.33M/1L)(342.3g/Mol)(1L/1000mL)(200mL/1) = 22.6g Sucrose



1. Added diH2O (approximately 125mL) to beaker and placed it on the stir plate with stir bar. Labeled beaker "50mM HEPES pH 7.5, 0.33M Sucrose."
2. Weighed out 2.383g HEPES and 22.59g (rounded up to 22.6g) Sucrose using weigh boats.
3. Added the HEPES, waited until it was completely dissolved, then added the sucrose slowly in small increments.
4. Once solute all completely dissolved, we calibrated the pH meter using the fresh standards.
5. pHd using NaOH and transfer pipette.
6. Transferred to a graduated cylinder. Rinsed beaker by adding diH2O to beaker, then used that water to BTV in the graduated cylinder at 200mL. 
7. Transferred to a 250mL glass bottle and completed rest of label. 
8. Stored in 4 degrees C refrigerator.
9. 

Observations and Results: 



We noticed that the solute dissolved evenly when adding slowly so we didn't overwhelm the solvent. Also, it took approximately 3 transfer pipettes to pH to 7.5 using the NaOH.





Conclusion: 



Making solutions is FUN!





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