LaCava Research Wiki

100 mg cell powder extracted at 1:4 w/v with affinity capture monitored by western and staining

A typical starting scale is 100mg

• ​after initial testing, scale up may be needed for other analytical approaches - the protocol scales with roughly equivalent results to ~0.5g but an increase in background may be observed as scale increases (see other entries). 
• A typical initial extraction regime includes 3 standard NaCl concentrations (100, 300, 500 mM), 20mM HEPES pH 7.4, and 0.5% v/v Triton x-100 used at 1:4 or 1:5 w:v
• The following fractions will be monitored:

1. Input (clarified lysate) - monitors soluble fraction in the given buffer.
2. Flow through (what did not bind to the affinity resin) - of the soluble fraction, what was depleted?
3. Pellet - what fraction of the protein was insoluble in the given buffer?​
4. Elution - how much protein was captured
   

A common result is that, for certain proteins such as those in membranes or chromatin, more protein is released to the extract at high salt. Depending on the tag, higher salt may result in a decrease in the efficiency of depletion however - tags such as 3xFLAG, GFP, and SpA are effective to at least 0.5M NaCl.

1. Prepare the buffers to be used - 100, 300, 500 mM NaCl in 20mM HEPES pH 7.4 and 0.5% v/v Triton x-100​

• ​some of this buffer will be placed at RT to be used for extracting powder (protease inhibitors will be added to this)
• the rest of the buffer will be held on ice for subsequent washing
• use Roche EDTA-free protease inhibitor tablets which can be prepared as a 100x stock in water (this can be frozen)
• ​these tablets come in two sizes - mini tablets are 100x in 100ul, normal tablets are 100x in 500ul

1. Dispense 100 mg of powered to three tubes (100, 300, 500) for each cell-type to be tested - hold on N2
2. Prepare the as much buffer as needed, plus one, and add protease inhibitors to 1x - hold at RT
3. Move tubes to rack at RT ~1 min
4. Extraction @ 1:4 w:v

• use 1:5 or 1:9 if the cell material is produces a large degree of lipids and/or pellet which will be observed after centrifugation - otherwise working concentrated is always advised. pH control can be an issue, extracts should be tested that the pH is that expected - otherwise the concentration of the buffering agent should be increased.
• Add 400 ul RT buffer to each tube of powder - vortex to resuspend fully - typically ~15 sec - hold on ice. a clump(s) of aggregated 'membranes' may be seen, but should otherwise appear as homogenous cloudy extract

1. ​Sonicate 1-2x for 2 sec, setting 2-3 (low power), repeat if needed - at 100mg 2x should be enough - treat all tubes the same

• position probe to center, ~2/3 - 3/4 deep into the sample
• power setting should be chosen to minimize foaming (use the lowest that works in 1 - 2, 2 sec pulses)
• only do as many rounds as needed to disperse aggregates, typically 1 - 2 rounds at this scale - if any tube needs an extra pulse, give all tubes the same treatment
• ​keep tubes on ice between treatments
• examine tubes after each round to assess the degree of success - should appear cloudy but clump free - homogenous "crude extract." 
  

1. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)

• Transfer cleared extract to fresh tubes; save ~5% (v/w+v) - 26ul / 500 - of the lysate as “Input” 
• ​combine with 10ul of 4x LDS - may be frozen - 4ul 10x reducing agent DTT will be added before running the gel; final vol = 40ul
• load 8ul of this 40ul soln on a Western, ~1%
• the amount used depend on the level of expression of the protein - 0.4% has been used for high expressers

1. Save the pellets @-20°C to be extract with 400ul of 1.1x LDS @ 70°C with mixing (5’) 

• ​Add 260ul of H2O and 100ul of 4x LDS
• results in 360ul of 1.1x soln - may be frozen and extracted later.
• add 40ul 10x reducing agent before extraction, giving 400ul final.
• load 5ul for Western, ~1%

1. Add cleared extract to equivalent of 10ul of pre-equilibrated affinity medium slurry 

• pre-wash beads with appropriate buffers - 3 x 1ml
• Add clarified extract to tubes containing beads

1. Incubate @ 4°C for 30’
2. Remove depleted extract

• save ~5% (26ul; treated as Input, above) of the flow through “FT” (load 8ul in 1x LDS for Western, ~1%)

1. Wash beads 3x 1ml extraction buffer

• switch to fresh tubes during 2nd wash (reduces background from non-specific binding of material to tubes released at elution)

1. Elute with 18ul of 1.1x LDS, 70°C for 5’ with shaking
2. Transfer the eluate to fresh tubes (“Elution”)

• save 2ul of the eluate (10%) for Western, use the rest for stained gel​

1. Ensure each sample is in 1x LDS and with (1x) 50mM DTT
2. Heat all samples @ 70°C for 10’

• extracted pellets were already heated to 70*C with 50mM DTT - they are good to go.

1. ​Load and run gel - ~200v for ~1hr on a NuPAGE Bis-Tris w/ MOPS buffer

​:*prepare 50 and 200ng BSA standards to be loaded on gel for total protein staining

One gel - with 90% of the elution in LDS - will be subjected to total protein staining - protocol depends on stain The second gel - with the input, FT, pellet, and 10% elutions - will be for Western, see below

1. Wet transfer 70V for 1.5h
2. Blocking: TBST/5% milk, RT, 2hrs
3. wash w/ TBST - until liquid is clear - no cloudiness from milk remains
4. Primary Ab - determined per antibody, 2hr incubation at RT or 4*C overnight
5. wash 3 x 10' w/ 50ml TBST
6. Secondary Ab - determined per antibody - typically 1:10,000 for HRP conjugated used in ECL - RT, 1hr
7. wash 3 x 10' w/ 50ml TBST
8. Add ECL reagent (Luminata Forte Western HRP Substrate, Millipore, Cat. No. WBLUF0100)