TITLE: Brooks Lab Western Blotting Protocol
AUTHORS: Brooks Lab University of California, Eva Robinson, Alison Tang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a Western Blotting Protocol for Brooks Lab, Department of Biomolecular Engineering, University of California,  Santa Cruz.</div></div>

[STEPS]
?. [RIPA Lysis Buffer]
Make , cover, and freeze in 10 ml aliquots at .  ORUse pre-made Pierce RIPA buffer, 100 ml, which is aliquoted in  aliquots and stored in
[RIPA w/o protease inhibitor]
-20 °C
10 ml
-20 °C
?. [RIPA Lysis Buffer]
Before use, thaw and add 1 tablet of protease inhibitor (PI) per 10 ml aliquot. (Roche cOmplete™, Mini Protease Inhibitor Cocktail).
?. [Harvesting Cells and Preparing Lysate]
Wash confluent 10 cm plate of cells 2X in ice cold PBS.
Always keep everything on ice, unless otherwise indicated.
?. [Harvesting Cells and Preparing Lysate]
Add  to cells, and scrape the cells to remove them from the dish. Transfer to a pre-chilled 2 ml tube .
[cold RIPA with PI]
on ice
?. [Harvesting Cells and Preparing Lysate]
Incubate , with periodic vortexing.
on ice
?. [Harvesting Cells and Preparing Lysate]
Pellet the insoluble material by spinning at max speed in refrigerated () microcentrifuge .
4 °C
?. [Harvesting Cells and Preparing Lysate]
Transfer supernatant to a clean LoBind Protein 1.5 ml tube as lysate. To avoid multiple freeze-thaw cycles, make aliquots (generally  each).
200 µl
Additional Sonicator or BioEruptor sonication step often needed here for more complete nucleic acid removal. See below.
?. [Harvesting Cells and Preparing Lysate]
Store lysates aliquots at .
-80 °C
?. [Sonication of Lysate to break up nucleic acids]
Place samples in a 1.5 ml LoBind Protein microfuge tube and prepare a volume balance tube.
?. [Sonication of Lysate to break up nucleic acids]
Bring a timer, ear cuffs,   samples, and balance to second floor to Kamakaka Lab to get the plastic tube adaptor (in back of second drawer in 2nd bay from the back of the lab.)
on ice
?. [Sonication of Lysate to break up nucleic acids]
Sonicator is in cold room down the hall. Put the outflow tube up on the shelf and fill glass reservoir with water to top of white line.
?. [Sonication of Lysate to break up nucleic acids]
Sonicator settings: timer on hold, use max setting (10), constant, then flip on.
?. [Sonication of Lysate to break up nucleic acids]
Hold tubes in the adaptor in the sonicating water bath for .
?. [Sonication of Lysate to break up nucleic acids]
Place tubes  to cool for .
on ice
?. [Sonication of Lysate to break up nucleic acids]
Repeat the 30’ sonication (= total of two rounds on max at .)
?. [BCA Assay for Protein Concentration Determination]
Pierce BCA Protein Assay Kit #23225 or #23227, Thermo Sci with 2 mg/ml BSA standard.Use instructions from kit. Equilibrate reagents, samples, and standards to . Use the Microplate reader instructions.
-20 Room temperature
?. [BCA Assay for Protein Concentration Determination]
Prepare a dilution series of BSA in working range of  –  from 1 glass vial of stock 2 mg/ml BSA in the kit for the standard curve:  ABCD1VialRIPA buffer, μl2 mg/ml BSA or standard, μlFinal con μg/ml2A0300 of stock20003B125375 of stock15004C325325 of stock10005D175175 of B7506E325325 of C5007F325325 of E2508G325325 of F1259H400100 of G2510I40000
ABCD1VialRIPA buffer, μl2 mg/ml BSA or standard, μlFinal con μg/ml2A0300 of stock20003B125375 of stock15004C325325 of stock10005D175175 of B7506E325325 of C5007F325325 of E2508G325325 of F1259H400100 of G2510I40000
?. [BCA Assay for Protein Concentration Determination]
Prepare fresh working reagent 50:1 of A:B, enough for all standards, unknowns, and replicates of the unknowns (should have n=3).
?. [BCA Assay for Protein Concentration Determination]
Add  of unknown or standard to 96-well plate well followed by  using 200 μl pipettor and gently mix avoiding spillover.
25 µl
[working reagent]
?. [BCA Assay for Protein Concentration Determination]
Cover plate with foil and incubate  for  in shaking incubator, gently shaking.
37 °C
?. [BCA Assay for Protein Concentration Determination]
Cool to .
37 Room temperature
?. [BCA Assay for Protein Concentration Determination]
Measure Absorbance on the VarioSkan at 562 nm.
?. [BCA Assay for Protein Concentration Determination]
Determine protein concentration of your unknown from the standard curve (the Skanit software will plot the curve and the unknowns on it if you edit the standards in the plate layout to include the concentrations above in the table.)
?. [SDS PAGE]
Prepare the following solutions for SDS-PAGE, Transfer, and Antibody Incubations:  AB1Running Buffer (Tris-Glycine)2100 ml10x TGX running buffer3900 mldd water  AB1Transfer Buffer2200ml5x Biorad Transfer Buffer3200ml 190 proof ethanol4600mldd water  AB1PBST250 ml10x PBS30.5 mlTween-204bring vol to 500 ml with dd water  AB15 % Milk Block (prepare immediately before use!)25 gSafeway brand non-fat milk pouch3100 mlPBST
AB1Running Buffer (Tris-Glycine)2100 ml10x TGX running buffer3900 mldd water
AB1Transfer Buffer2200ml5x Biorad Transfer Buffer3200ml 190 proof ethanol4600mldd water
AB1PBST250 ml10x PBS30.5 mlTween-204bring vol to 500 ml with dd water
AB15 % Milk Block (prepare immediately before use!)25 gSafeway brand non-fat milk pouch3100 mlPBST
?. [SDS PAGE: Denaturing samples in Laemmli reducing buffer]
In a fume hood, Add  to  to make MLB.
[2-mercaptoethanol]
[4x Laemmli sample buffer]
?. [Semi-dry Transfer to PVDF Membrane]
Pre-soak blotting stacks in 1X transfer buffer made according to directions on the bottle.
?. [Semi-dry Transfer to PVDF Membrane]
Pre-wet PVDF membrane from Biorad Transblot kit in 190 proof ethanol, then in transfer buffer.
?. [Semi-dry Transfer to PVDF Membrane]
Layer into Transblot drawer (stack, membrane, gel, second stack) before rolling out bubbles gently with conical tube
?. [Semi-dry Transfer to PVDF Membrane]
Run the transfer on Biorad setting, standard Mininigel TGX, 25mA, 25V, .
?. [Semi-dry Transfer to PVDF Membrane]
While running, pour 5% milk block in 1X PBST directly into cleaned small black Western blot box.
?. [Semi-dry Transfer to PVDF Membrane]
Incubate transferred blot for  at  in 5% milk block in 1X PBST.
95 Room temperature
?. [Semi-dry Transfer to PVDF Membrane]
Photograph the blot after the transfer to help keep track of orientation.
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Primary and secondary antibody bindingβ-Actin (C4): sc-47778ECL SuperSignal West Pico Chemiluminescent SubstrateUse 1:500 Dilutions for primary antibodies, in 5% milk/PBST
Note: Primary antibody (unconjugated with HRP) can be re-used 3x if kept frozen.
?. [Antibody Incubations and ECL visualization of target protein bands: Actin staining with anti-actin-HRP primary]
Actin staining with anti-actin-HRP primaryWash blot 3x with 1X PBST .
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab)Place blot in between sheet protector plastic and transport to imager in cassette to keep dark.
?. [BCA Assay for Protein Concentration Determination]
Export report as an excel doc.
?. [SDS PAGE: Denaturing samples in Laemmli reducing buffer]
Dilute samples with MLB at a ratio of 3 parts sample to 1 part MLB. So for  final volume, add   to  in 1.5 ml LoBind safety lock tube.
30 µl
[MLB]
[sample in RIPA]
?. [SDS PAGE: Denaturing samples in Laemmli reducing buffer]
Denature at  for .
95 °C
?. [SDS PAGE: Denaturing samples in Laemmli reducing buffer]
Transfer to ice.
?. [SDS PAGE: Loading and Running Gel]
Remove gel from package, take off green strip at the bottom, and rinse the wells three times with about  (use 1 ml pipettor).
[running buffer]
?. [SDS PAGE: Loading and Running Gel]
Place gel, tall plate facing out in outer side of a holder and the reservoir block at the other to make a running buffer reservoir. Put the other gel holder in place to take up space.
?. [SDS PAGE: Loading and Running Gel]
Fill the chamber to the 2-gel mark. Make sure there are no leaks before testing the circuit. Test the circuit by checking for bubbles after turning on power to 70 volts.
?. [SDS PAGE: Loading and Running Gel]
Using a 20 ul conte-tipped pipettor, load  of the samples in MLB onto gel, as well as  of Biorad Precision Plus protein ladder Dual Color.
30 µl
10 µl
?. [SDS PAGE: Loading and Running Gel]
Run at 100 – 120V, depending on desired resolution, until adequate separation of ladder lanes in sizes regions of interest.
?. [SDS PAGE: Loading and Running Gel]
Photograph the gel after the run with your phone to help keep track of orientation.
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Drain milk block from blots and add primary Ab (in milk block).
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Incubate in cold room  covered and shaking on orbital shaker, covered with plastic.
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
The next day, wash blot 3x/ in PBST.
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Freshly prepare secondary antibody at 1:10,000 dilution ( to ).
2 µl
[5% milk/PBST]
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Incubate in secondary antibody for , .
95 Room temperature
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Discard secondary and wash blot 3x/ in 1xPBST.
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Discard secondary and in the same box or moving the blot to a new box, add ECL working solution ( of both reagents mixed in a  conical and applied to blot.)
1 ml
15 ml
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Incubate in ECL for , shaking occasionally by hand, covered.
?. [Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding]
Drain blot and place between plastic sheets for imaging using Biorad GelDoc, as below. After imaging, the blot can be probed for actin as a protein loading control without stripping the blot, using anti-actin-HRP as described below.
?. [Antibody Incubations and ECL visualization of target protein bands: Actin staining with anti-actin-HRP primary]
Incubate ,  in the primary actin antibody 1:500 freshly made dilution in 5% Milk block.
95 Room temperature
?. [Antibody Incubations and ECL visualization of target protein bands: Actin staining with anti-actin-HRP primary]
Wash 2 – 3x in 1x PBST  each.
?. [Antibody Incubations and ECL visualization of target protein bands: Actin staining with anti-actin-HRP primary]
Incubate in ECL as above (step 51) and image.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Place blot on white screen.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Place screen into chamber drawer.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Select new protocol, then blot, then chem hi sensitivity and position blot under live focus setting.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Under Applications, select chemidoc hi sensitivity to photograph target bands. Note the image size, the gain (2X), and bin (2x2) to make sure the image size is the same when the protein standards are photographed in order to be able to merge the images.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Under Live Acquire, select acquisition settings: 1, 600, for range and total =100 acquisitions (the 100 can be lowered for longer exposure), starting at 0.25 start time for high expression target.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Freeze and save the image of your choice as it comes up.
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Without moving the blot, take a picture to visualize the protein standards by selecting Custom under Applications, then create an Epi illumination protocol with the same gain and binning as used for photographing the bands (double check the image size prediction under the options…)
?. [Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab) ]
Merge the target bands and the protein standards pictures in Image Lab software using Image tools.
Note: Imaged blots can be stored at 4ºC in PBST for stripping and re-probing. Some folks also store them frozen in -20ºC flat in plastic bag.