5-min Protein Prep for SDS-PAGE Western Blot
Before you start you should know the expected size of your protein (for western blot). You will use this information to determine 1) what percent gel you’ll need to use 2) which strain should be your positive control, 3) which molecular weight standard you will use, and 4) the appropriate antibodies for your loading control (if you’re using one).
Also note that you should quickly freeze/thaw your samples (i.e. store preps in -80⁰C and thaw in 95⁰C). The Krafft point for SDS is around 16⁰C, well before the freezing point of 10% glycerol (-1.6⁰C). When the temperature of your sample is between 16⁰C and -1.6⁰C, proteases will have an opportunity to refold and degrade proteins (including yours). By quickly freezing and thawing your sample you minimize the potential for degradation.
Culture prep
• Collect 500 µl of O/N culture. (May need to collect more depending on culture thickness.)
• Centrifuge 3min at 3.0 rpm
• Wash in 500 µl H2O
• Centrifuge 3min at 3.0 rpm
• Discard supernatant. Re-suspend in 1ml of respective media and inoculate into:
• For Yeast- in a culture tube, sub into 3ml of media (for a total of 4ml in culture tube) & incubate at 30⁰C for ~4hr. (want log phase growth i.e. budding)
• For Hyphae- in a 250mL flask, sub into 9 ml of media with 10% serum [for a total of 10ml in flask 8ml media + 1ml serum + 1ml resuspended cells] & incubate at 37⁰C for ~4hr. (want log phase growth i.e. lots of hyphae)
Rapid protein prep
FOR YEAST CULTURES:
Collect approximately 1e8 cells:
• Visually inspect cultures.
• If they look hyphal or pseudohyphal your spec reading will be off.
• If all cultures are approximately of equal density you may choose one culture that appears to be the least dense and use that sample to represent the others (variation will be accounted for later). If you choose to do this remember to record the sample that you chose.
• Collect and record OD600 reading for cultures
• In a 0.5ml microfuge tube, dilute 2µL of resp. media into 198µL of water (blank)
• In a second tube, dilute 2µL of subculture into 198 µL of water (sample)
• Calculate amount of sub culture to collect
• (ABS OD600 Reading)*100= # of 1 OD600 /ml
• 1 OD600 = 1e7 cells/ml , therefore your (OD600 Reading)*100*1e7=___e7cells /ml
• (___e7cells /1 ml) = (1e8 cells/χ ml) solve for χ
• χ = # ml’s required to obtain 1e8cells
• Collect the required volume of each culture for 1e8 cells (may collect more if low-abundance protein)
• Centrifuge 3min at 3.0 rpm
• Discard supernatant
• Remove as much excess media as possible
• Resuspend pellet in 50µL of 4% SDS ULSB +20mM DTT
• Heat at 90-100°C for 5min *** remember to use lid locks***
• Centrifuge at max speed for at least 5 min (longer may prove helpful, but not always necessary)
• Your protein is in the supernatant. As long as you are careful not to disturb the pellet, no need to separate the two.
• Collect and record A280 readings for protein preps
• In a 0.5ml microfuge tube, dilute 2µL of 4% SDS ULSB +20mM DTT into 198µL of water (blank)
• For each respective prep, dilute 2µL of prep into 198µL of water (sample)
• Normalize the A280 units (aim for ~25-50 A280)
• Calculation: [(A280 Reading) x 100 x (volume supernatant left after spec)]/(desired units)
• Example: A280 Reading = .414 ; I want to normalize to 35 A280 units; and I started with 50µL supernatant… so, I calculate [.414 x 100 x 48] / 35 = 56.8
• 56.8µL is final volume. So, to calculate how much more 4% SDS ULSB +20mM DTT to add to normalize, need to subtract initial volume from final volume:
vf – vi = 56.8 – 48 = 8.8µL to add to tube to dilute protein.
FOR HYPHAE:
Collect entire 10 ml culture in 15 ml conical.
• Centrifuge for 5 min at 1000rpm in Berman lab centrifuge (swinging bucket).
• Discard supernatant
• Collect and transfer the hyphal cells to a 1.5 microfuge tube
• Cut the tip off of a 1 mL pipette tip to re-suspend in 500 µL H2O and transfer into 1.5 ml microfuge tube.
• Centrifuge 3 min at 3.0 rpm
• Discard supernatant
• Remove as much excess water as possible
• Resuspend in 100µL of 4% SDS ULSB +20mM DTT
• Heat at 90-100C for 5min *** remember to use lid locks***
• Pellet (Max speed)
• Collect supernatant ( this step can be omitted)
• Collect and record A280 readings for protein preps
• In a 0.5ml microfuge tube, dilute 2µL of 4% SDS ULSB +20mM DTT into 198µL of water (blank)
• For each respective prep, dilute 2µL of prep into 198µL of water (sample)
• Normalize the A280 units (aim for ~25-50 A280)
• Same calculation/procedure to normalize as above
SDS-PAGE
• Load desired A280 units per well of sample (once normalized, 10µL of prep should be sufficient; if less than 25 A280 units in prep, try loading a larger volume)
• Also load 10 µL of the molecular weight standard
• Run gel @ 30V for 15 min then @200V for ~45 min (or till dye front is at bottom of gel ***try not to run the dye front off!)
TRANSFER FOR WESTERN
TRANSFERING PROTEINS FROM GEL TO BLOT:
• Cut PVDF blotting membrane and blotting paper to size of gel.
• Wet membrane first with “dry” methanol; pour off back into “dry” methanol bottle.
• Rinse blot in milliQH2O water until water no longer beads off.
• Set up “sandwich” and transfer apparatus as described on pg. 4-7 in Mini Trans-Blot Electrophorectic Transfer Cell Manual. Be sure to pay attention to alignment of gel and blot.
• Run transfer for 1hr at 100V –OR– overnight (12hrs) at 30V.
CHECKING TO ENSURE PROTEIN TRANSFER:
PONCEAU Membrane
• Take apart transfer.
• Rinse membrane in milliQH2O and put in blotter paper sandwich to dry. Can dry overnight or in 37⁰C for 5-10 min.
• Re-wet blot with “dry” methanol; pour off back into “dry” methanol bottle.
• Rinse membrane in milliQH2O and add enough Ponceau stain to cover the membrane.
• Shake at room temp for a short amount of time. If protein bands seen = Good. This means transfer occurred.
• Mark (with a pencil) the molecular weight marker on the membrane.
• Rinse membrane in milliQH2O and then with “wet” methanol; pour methanol off back into “wet” methanol bottle.
• Rinse membrane again in milliQH2O and put in blotter paper sandwich to dry. Can dry overnight or in 37⁰C for 5-10 min.
COOMASSIE Gel
• After transfer, put gel in tupperware container with enough Coomassie stain to cover the gel.
• Shake for ~20min at room temp.
• Rinse membrane with Coomassie Destain.
• If gel shows clear (blue) = Good. This means all your protein transferred! If you have dark bands, not good...
DEVELOPING THE BLOT
• Rewet blot with “dry” methanol; pour off back into “dry” methanol bottle.
• Rinse with milliQH2O and pour H2O off.
• MILK BLOCK: Add ~20ml 2% milk solution to blot in tupperware container and shake for 30min at room temp.
• Pour off milk block and rinse briefly in ~20ml 1X TBST.
• 1⁰ antibody Add ~20ml 0.2% milk solution to blot and add ~10µL 1⁰ antibody (MS∞GFP or other). Swirl, then shake for 1hr at room temp, or for up to 12hrs in cold room.
• Pour off 1⁰ antibody solution.
• Rinse 3x quick and 3x (5min) in 1X TBST.
• 2⁰ antibody Dilute 3µL 2⁰ antibody (Goat∞MS HRP or other) into 300µL 0.2% milk solution. Add 40µL of this dilution to ~20mL 0.2% milk solution in tupperware container covering blot. Swirl, then shake for 1hr. at room temp.
• Pour off 2⁰ antibody solution.
• Rinse 3x quick and 3x (5min) in 1X TBST.
• Leave in small about of TBST while setting up to development materials.
• Mix 700µL of each part of chemiluminescent reagent (Supersignal Fempto) together in microfuge tube (for a total of 1400µL).
• Cut a clean piece of transparency in half. Wet one half with ddH2O and lay on clean piece of saran wrap.
• Dispense half of chemiluminescent mixture in the center of transparency where you will place blot.
• Place blot on top of drops and add the rest of the chemiluminescent on top of the blot.
• Wet second half of transparency with ddH2O, and overlay this on top of blot
• Smooth out bubbles, and spread excess reagent on top of transparency.
• Fold saran wrap over transparency and fold edges to keep reagent from leaking out.
• Make sure outside of saran wrap is not wet with chemiluminescent mixture, because this will cause film to become highly overexposed.
TO VISUALIZE DEVELOPED BLOT
On Chemiimager:
• Place black tray in upper slot in chemiimager and place sandwich on top.
• Turn filter to 1 on imager.
• Expose for 5-30 min.
In film cassette:
• Dark rooms located on 5th and 7th floors in MCB.
• Place sandwich in exposure cassette with film.
• Expose for anywhere from a few seconds to overnight, depending on protein abundance and signal intensity.
• Develop film.
RECIPES
For 10mL 4% ULSB:
3.6g Urea (for a final 6M conc)
~4.8mL milliQH2O
1mL 20% SDS
2mL 5x Lammeli Sample Buffer
Weigh out 3.6g Urea FIRST, add H2O second (start with 3.5mL), warm to bring urea into solution, add SDS while warming, then add LSB. Fill to 10mL with ddH2O. Make 1.5 mL aliquots and freeze at -20⁰C.
Freezer Stocks of DTT = 1M
For 4% ULSB + 20mM DTT:
Add 30µL 1M DTT to 1.5mL 4% ULSB. Mix well.
If using a Loading Control:
1° Antibody: Anti-tubulin conc. = 1:5000
Add 4µl antibody to 20mL 0.2%milk
2° Antibody: HRP∞Rat conc. = 1:10000
Add 2µl antibody to 20mL 0.2%milk
200mL methanol
700mL milliQH2O
100mL 10X transfer buffer
1mL 20% SDS.
Add reagents in the order listed. Mix well.
Ponceau
2g Ponceau
150mL 20% TCA (trichloroacetic acid)
Bring to 1L with milliQH2O
Coomassie
1.25g Coomassie
1.25L Methanol
250µL acetic acid
1.0L milliQH2O
Mix well, then filter sterilize.
Broad Molecular Weight Marker
Make aliquots of 100µL:
5µL 20X Broad Range Marker
95µL 4% ULSB + 20mM DTT
Add BRM to ULSB+DTT, flick tube to mix, and boil 3 min at 95⁰C. Store in -20⁰C freezer.
For 1 Liter 1X TBST
Add 100mL 10X TBST to 900µL milliQH2O. Mix well.
2% Milk Solution
Measure out 1g dry milk powder in a 50mL conical tube.
Add 50mL 1X TBST, and mix well.
0.2% Milk Solution
Add 30mL 2% milk solution to 270mL 1X TBST. Mix well.