TITLE: Differential extraction of detergent-insoluble protein aggregates from cultured mammalian cells - a benchtop centrifuge method
AUTHORS: S. BERGINK, W. HUITING

[DESCRIPTION]
Protein aggregation is strongly associated with accelerated ageing and (neuro)degeneration, and considerable effort is directed towards studying this process in vivo. Reliable, unbiased quantification and analysis of endogenous, insoluble protein aggregates requires their specific isolation from the total proteome. Here, we describe a user-friendly differential detergent fractionation method to sequentially extract increasingly insoluble proteins from cultured cells. We also discuss options for downstream quantification and analysis. The use of a benchtop centrifuge instead of ultracentrifugation makes this method simple and cost-effective.

[STEPS]
SECTION: Fractionation
8. Take desired volume from supernatant to serve as Igepal soluble fraction (fraction I)
Add 4XSB into supernatant. Mix by brief vortexing.
Boil (5 min, 95°C).
Store samples at -20°C.
SECTION: Fractionation
1. Prepare cell culture starting material according to your research question. 
a.     Harvest cells via scraping in ice-cold PBS.
b.     Pellet cells in 1.5 microcentrifuge tubes via low-speed centrifugation in a pre-cooled benchtop centrifuge (5 minutes, 500 x g, 4 °C).
c.     Discard supernatant.
SECTION: Fractionation
2. Prepare assay buffers as needed. 
a.     Prepare buffer A.                                              
    i.     Prepare a minimum of 120 ml + (100 ml per 500 mg expected protein yield from lysis) buffer A per sample.            Example: for one well of a 6-well culture plate, with HEK293t cells at 90% confluency, the expected total protein yield after lysis would be 400-500 mg. If three samples will be generated from one well each, prepare 3 x (120 + 100) = 660 ml of buffer A. 

b.     From buffer A, prepare buffers EB, I and S.                                               
    i.     Prepare 550 ml wash buffer EB per sample.
    ii.     Prepare 200 ml of both buffer I and buffer S per sample.

c.     Prepare buffer U.
SECTION: Fractionation
3. Lyse pelleted cells in 1.5 ml microcentrifuge tubes in 150 ml ice-cold buffer I.
a.     Vortex cell pellets in buffer I until dissolved completely, then put tubes on ice.
b.     Vortex cell pellets 5-10 seconds every 10 minutes for 30 minutes.
c.     Homogenize cell lysates further with a 1 ml syringe and 30G needle (5 strokes). 
d.     Vortex homogenates 5-10 seconds, leave on ice for another 10 minutes.
e.     Visually verify that cell lysis is complete (no cell clumps left).
SECTION: Fractionation
4. Perform protein measurement, preferably using a detergent compatible protein quantification method (e.g. DC protein assay, Bio-Rad) and performing measurements in triplicate.
SECTION: Fractionation
5. Equalize protein concentration between samples using remaining buffer I. Equalize to ~250 mg of total protein per sample.
SECTION: Fractionation
6. Take desired volume from the equalized homogenate to serve as whole cell lysate (fraction WC, i.e. loading control)
a.     Take 15-25 mg for simple assay verification (e.g. staining, Western blotting), take more for multiple downstream analyses.
b.     Add 4XSB into homogenate. Mix by brief vortexing.
c.     Boil (5 min, 95 °C).
d.     Store at -20 °C.
SECTION: Fractionation
7. Centrifuge remaining homogenate in a pre-cooled (4 °C) benchtop centrifuge at 14,000 x g for 45 minutes.
SECTION: Fractionation
13. Take desired volume from supernatant to serve as SDS soluble fraction (fraction S).
a.    Add 4XSB into supernatant.
b.    Mix by brief vortexing.
c.    Boil (5 min, 95 °C).
d.    Store samples at -20 °C.
SECTION: Fractionation
9. Carefully take off the rest of the supernatant, discard.
SECTION: Fractionation
10. Wash the protein pellet.
a.     Add 250 ml ice-cold wash buffer EB without disturbing the protein pellet.
b.     Centrifuge in a pre-cooled (4 °C) benchtop centrifuge at 14,000 x g for 15 minutes.
c.     Take off the supernatant, discard.
SECTION: Fractionation
11. Dissolve the 1% igepal-CA630 protein pellet in buffer S.
a.     Add appropriate volume of buffer S (volumeS = volumestart - volumeWC - volumeI) at room temperature.
b.     Carefully break up the protein pellet using a p200 pipettip.
c.     Put the tube in a dry block heating shaker at room temperature, shaking at 1000-1200 rpm for 1 hour.
d.     Check if protein pellets are completely dissolved. If not, extend shaking time.
SECTION: Fractionation
12. Centrifuge the protein suspension in a benchtop centrifuge at 14,000 x g for 45 minutes at room temperature.
SECTION: Fractionation
14. Carefully take off the rest of the supernatant, discard.
SECTION: Fractionation
15. Wash pellet.
a.     Add 250 ml wash buffer EB (room temperature) without disturbing the protein pellet.
b.     Centrifuge in a benchtop centrifuge at room temperature, 14,000 x g for 15 minutes.
c.     Take off supernatant, discard.
SECTION: Fractionation
16. Dissolve the 1% SDS insoluble protein pellet in buffer U
a.     Add 35-50 ml buffer U to the protein pellets to ensure proper dissolution.
b.     Put the tube in a dry block heating shaker at room temperature, shaking at 1000-1200 rpm for 3 hours to         overnight.
c.     Check if pellet has dissolved properly. If not, extend shaking time.
d.     Freeze samples in liquid nitrogen and store at -80 °C.

Note:Avoid heat greater than 25°C when dissolving proteins in buffer U.
SECTION: Downstream quantification and analysis
17. Run WC, I and S fractions in a polyacrylamide gel.
a.     Load 3-5 ug of protein per sample. This is generally sufficient to obtain a good signal/noise ratio in staining or stain-free quantification.
SECTION: Downstream quantification and analysis
18. Run U fractions (SDS insoluble proteins) separately in a polyacrylamide gel
a.    Add 1 ml of 0.5% bromophenol blue to each sample to make loading easier
b.    Vortex, and briefly centrifuge
c.    Load 25-50% of fraction U.
SECTION: Downstream quantification and analysis
19. After separation, stain protein fractions using a method of your choice.
a.     For simple analysis, we recommend colloidal Coomassie (G-250) staining for the WC, S and I fractions. This offers user-friendliness, good sensitivity and reproducibility. 
b.    Fraction U generally contains very few proteins, so we recommend a highly sensitive Silver-staining
SECTION: Downstream quantification and analysis
20. Quantify stained protein fractions
a.     Use imaging software to plot and measure whole lane intensities (e.g. with the freely available package ImageJ) to obtain a relative quantification between fractions.
b.     When unstained ladder was included, normalize lane intensities to ladder intensities to get an approximate absolute quantification.