Narla Lab Western SOP

TS 8-5-20
*When collecting cell pellets and preparing lysate, if looking at methyl-C, use CoIP buffer instead of RIPA
– these are both made in aliquots in the freezer. Add based on pellet size, about 60-70uL per pellet, but
if larger pellet then add 100uL.

Protein Quantification
Reagents:
Water (Molecular Biograde (MBG) or MilliQ filtered water)
15mL or 50mL conical tube wrapped in foil
96 well plate
Pierce BCA Protein kit (Reagent A + B, Thermo Scientific, cat# 23228 and 1859078, respectively)
Eppendorf tubes
Samples on ice
BSA aliquot (20 mg/mL, from -20⁰C)

-There are always 18 standards (x9 into 2 wells each): 0, 2, 4, 6, 8, 10, 12, 16, 20

1. Thaw samples, and sonicate in cold room for 45s.

2. Prepare Master mix. Master mix/green calculation:

(18 + # of your samples) x 200uL = x/51 = amount of reagent B

uL amount of B x 50 = uL amount of A

For example, 18 samples  18+18 = 36  38 total samples, 200x38 = 7,600uL, 7,600uL/51 = 149uL
reagent B, so 149uL x 50 = 7,450uL of A – mix together in foil covered tube

3. Spin samples at max speed (on little centrifuge) for 15 min, and carefully pipette liquid into
newly labeled eppendorf tube (without disturbing the pellet which you will discard).
4. While spinning, prepare standards and add 195uL of A/B Master mix to each well of 96 well
plate.
- To prepare/dilute standards (other than the 0 is just 20uL of water and 20 does not need to be
diluted):

Standard # = amount of BSA aliquot to add, to make up total volume of 20uL of each standard.
For example, for 2, add 2uL of BSA stock to 18uL of water, for 4 add 4uL of BSA stock to 16uL of
water, etc).
5. Add 5uL of standard or protein sample into each well, pipetting up and down to mix.
6. Incubate for 30 minutes at 37⁰C (in shaker incubator).
7. Read plate at 562nm on plate reader.
8. Use spreadsheet to calculate protein concentrations, and prepare aliquots of:
a. Protein (40-60ug, for methyl-C no less than 45ug)
b. Water
 c. Loading dye
d. DTT 1:10 total volume (stock is at 1M in 10uL – add 90uL of water before using, and
throw away any remaining)
-if not running same day, add DTT right before running/next day
9. Spin at 8,000rpm for 2 minutes. Can freeze now in -20⁰C if not running.
10. Boil for 10 min with stopper caps (so tubes don’t pop open when boiling).

Running Gels
1. Get appropriate % and lane # gel from fridge, and REMEMBER TO REMOVE GREEN TAPE FROM
BOTTOM. Remove comb.
2. Make sure no leaks from cassette when gels closed in (place gels on both sides of cassette or use
buffer dam if only running 1 gel). Fill box/cassettes very full of running buffer (made in carboy),
to 2 gel line for only 2 gels, and 4 gel line for 4 gels, but always make sure cassettes are full
- Tall side of gel always facing out, so numbers are in order Left to Right.
- Make sure cassettes are pushed in all the way to bottom snuggly in box.
3. Add samples to lanes and 5uL of ladder (PageRuler Prestained, Thermo Scientific, cat# 26616,
from -20 common reagent box in 3411).
4. Red probe is on the left, and run at 300V, ONLY UNTIL green band from ladder hits the bottom –
only about 25 minutes to run.

Activate gels and Transfer

1. Crack open gels, and carefully cut off top fingers/lanes and bottom edges.
2. Put 2-5 (up to 10mL) of TBST (or PBST for methyl-C) onto Chemidoc tray, wet fingers with TBST
(or PBST), and carefully put gels on tray (2 at a time), ladder on left side.
3. Open “Image Lab” on computer and New Protocol  Gel imaging  under Application, choose
Protein and Stain free Gel, and 1 minute (is all that is needed) for Gel Activation. The Position
Gel – if doing 2 gels, change filter as prompted by moving the little black knob on side of
Chemidoc to position it specifies, and then zoom in or out to center gels on screen. Click “Run
Protocol” once gels are centered on image.
4. Get Trans Blot Turbo Pack from fridge (next to gels) – Midi for 2 gels and Mini for 1. Open pack
carefully, pull top white tab and put membrane with filter onto transfer pad. Squeegee gently
to get bubbles out, but not too much so don’t squeeze out transfer buffer liquid.
5. Put gels on Left to Right, with heavier bands out (ladder on the left), with gels straight even if
ladder isn’t, and they will transfer in towards each other.
6. Put other filter paper on top with tabs even/on top of each other, and carefully roll out any
bubbles.
7. Put lid of drawer on and lock in one smooth motion.
8. Put drawer in TransBlot Turbo machine, and select “List”  BioRad  select gel (Midi or Mini)
 Mixed MW, then Run (“A-Run” if only 1 drawer). Takes about 7 minutes to transfer.
9. Unlock and open lid, and pull up slowly – top filter should come off with lid. Carefully take gel
off membrane and put blots in TBST (or PBST).
10. To image blot, select under Application menu “Blots” Stain Free Blot, and click “Position Gel”
then open Chemidoc door and position membrane in the center, then click “Run Protocol”.
 11. Rinse transfer cassettes/drawer well with distilled water and clean off Chemidoc tray with
distilled water.

Block and Add Antibody

1. Block for 1 hour with 5%milk on shaker room temp (if looking at methyl-C, use 3% milk in PBST).
2. To cut to add appropriate antibodies, use cooling block and clear 96 well plate lid to line up
where to cut blot.
3. Add primary antibody in concentrations give in 5mL of 5% milk/TBST (for example, if 1:1000, add
5uL antibody into 5mL milk/TBST).
- Primary antibodies are in new and validated primary antibody box in fridge in 3411, and others are
alphabetized in 3411 freezer
4. Run on shaker overnight in cold room.
Next day:
5. Save primaries (except Vinculin) in labeled 15mL conical tubes.
6. Wash 3X for 10min each with TBST (or PBST).
7. Add secondary at 1:2500 in 5mL (so 2uL secondary into 5mL of milk/TBST) for 1 hour on shaker
room temp.
8. Wash 3X for 10 min each with TBST (or PBST).

Image on Chemidoc, then Strip/Add next antibody

1. Turn on about 10 minutes before use (to warm up).
2. Make Protoglow ECL (cat# CL-300) – add 1 mL of brown bottle (Reagent A) and 1 mL of clear
bottle and mix (they are located in the 3411 fridge, 3 rd shelf in door).
3. Add just enough of Protoglow mixture to cover the membrane, 5-10X using a pipettor.
4. Open ImageLab on the computer, click “New” protocol. Under “Application” select “Blots” 
“Chemi”.
5. Put blot in Chemidoc, make sure “No Filter” in “Filter position” that pops up by moving little
black lever on Chemidoc to far left to click (as shown on pop-up). Line up ladder straight (not
the membrane), and click Run protocol and do Auto. If it is taking too long (longer than 1
minute), do “Signal accumulation mode” and do a range of about 1-60s with 6 images to see
which timepoint gives you the clearest image/darker bands. Right click to “Save All” images
(save in folder labeled with experiment name, replicate number this is (n = #), and antibody.
a. Red means over-saturated.
6. When you have chosen your timepoint, select “Manual” to set the exposure time.
7. Then take a picture of ladder by selecting under Application menu  Blots  Colorimetric, and
switch back to check “The software will automatically optimize exposure time for intense
bands”.
8. If staining for more antibodies, strip by adding stripping buffer to cover blot, for 10 minutes 2X
on shaker room temp.
9. Wash with TBST (or PBST) for 10min 3X.
10. Add next primary antibody and shake overnight in cold room, then add secondary next day and
image again as described above.
*Always blot/image Vinculin last, and may need to dilute ECL with distilled water.