TITLE: Cellular protein extraction and Western blotting using dry transfer (iBlot system)
AUTHORS: Ralitsa R. Madsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A generic protein extraction and Western blotting protocol, allowing both for total cell lysis or cytoplasmic protein extraction only. The cytoplasmic extraction version has been used when Western blotting for PI3K and MAPK/ERK signalling components; total cell lysis with sonication has been used when Western blotting for histones. </div></div>

[STEPS]
?. [Cell lysis and protein extraction]
Pre-chill the benchtop microcentrifuge to  .Prepare cell scraper and a beaker with PBS - this will be used to rinse the cell scraper in between processing of different samples.
4 °C
?. [Cell lysis and protein extraction]
Aliquot 10 ml of stock lysis buffer and dissolve the required supplements that need to be added fresh (see "Guidelines" for buffer details). Keep on ice.
?. [Cell lysis and protein extraction]
Allow the snap-frozen cells to thaw on ice (one plate at a time, process each well for scraping and rinse the scraper in PBS in between individual samples).
?. [Cell lysis and protein extraction]
Add    of ice-cold lysis buffer per well and scrape the cells on ice.
150 µl
The volume can be reduced to 100 μl depending on expected yield. I use this volume when working with human pluripotent stem cells which give a high protein yield from a 6-well (3-5 mg/ml); this may not be the case with other cell lines and should be tested empirically.
?. [Cell lysis and protein extraction]
Transfer cell lysates to pre-labelled and pre-chilled tubes.
?. [Cell lysis and protein extraction]
Incubate for minimum   on ice.
?. [Cell lysis and protein extraction]
If performing nuclear lysis for extraction of histones: sonicate the samples on a Diagenode Bioruptor using 5 pulses of 3  ON,   OFF; setting =high. Following sonication, the lysates should appear clear/runny.
?. [Cell lysis and protein extraction]
Centrifuge the lysate at  and   for  .
4 °C
?. [Cell lysis and protein extraction]
Transfer the supernatant to new pre-chilled tubes and discard the pellets.
Stopping point: the samples can be stored at   at this point and processed for protein concentration measurements at a later time. Freeze-thawing should generally be limited, but up to 3 times has worked well for me in the past (alternatively, make multiple aliquots to avoid freeze-thawing of the entire volume each time).
?. [Cell lysis and protein extraction]
Dilute the samples for protein concentration measurements (usually 1:5 but will depend on the exact samples and the expected yield). Use molecular-grade H2O.
Remember to dilute the lysis buffer the same. It is easiest to perform these and all subsequent dilutions in PCR strip tubes.
?. [Cell lysis and protein extraction]
Prepare DC assay solution A+S according to the manufacturer's instructions (BioRad): use   Reagent S to   Reagent A (prepare a mastermix in excess of what you will need; e.g. for loading of 8 standards and 8 samples in quadruplicate = 25 * 16 * 4 * 1.2 (excess) = 1920 ~ 2 ml (2 ml Reagent A + 40 μl Reagent S).
20 µl
1 ml
?. [Cell lysis and protein extraction]
Add   of the A+S mix to each well of a 96-well plate for protein concentration measurements (e.g. Sterilin™ Clear Microtiter™ Plates; Thermo Scientific Sterilin 611F96 (for absorbance measurements) #11349163)
25 µl
?. [Cell lysis and protein extraction]
Add   of each sample or standard dilution in triplicate; use multichannel pipettor if dealing with multiple samples and all diluted in PCR strip tubes.Example layout  ABCDEFGHIJKLM1Plate 11234567891011122AS1 (0 mg/ml)S1S1G7_25G7_25G7_25Lysis buffer dilutionLysis buffer dilutionLysis buffer dilution3BS2 (0.125 mg/ml)S2S2G7_26G7_26G7_264CS3 (0.250 mg/ml)S3S3G7_27G7_27G7_275DS4 (0.5 mg/ml)S4S4G7_28G7_28G7_286ES5 (0.750 mg/ml)S5S5G7_29G7_29G7_297FS6 (1 mg/ml)S6S6G7_30G7_30G7_308GS7 (1.5 mg/ml)S7S7G7_31G7_31G7_319HS8 (2 mg/ml)S8S8G7_32G7_32G7_32
5 µl
ABCDEFGHIJKLM1Plate 11234567891011122AS1 (0 mg/ml)S1S1G7_25G7_25G7_25Lysis buffer dilutionLysis buffer dilutionLysis buffer dilution3BS2 (0.125 mg/ml)S2S2G7_26G7_26G7_264CS3 (0.250 mg/ml)S3S3G7_27G7_27G7_275DS4 (0.5 mg/ml)S4S4G7_28G7_28G7_286ES5 (0.750 mg/ml)S5S5G7_29G7_29G7_297FS6 (1 mg/ml)S6S6G7_30G7_30G7_308GS7 (1.5 mg/ml)S7S7G7_31G7_31G7_319HS8 (2 mg/ml)S8S8G7_32G7_32G7_32
?. [Cell lysis and protein extraction]
Add    of Reagent B to each well.
200 µl
?. [Cell lysis and protein extraction]
Wrap plate in foil and put on an orbital shaked at 300 rpm for  .
?. [Cell lysis and protein extraction]
Read 595 nm absorbance on a suitable plate reader.
?. [Cell lysis and protein extraction]
Calculate sample concentration based on the standard curve and adjusted for the applied dilution; remember to subtract the lysis buffer only blank.
?. [Cell lysis and protein extraction]
Calculate the amount of sample needed for loading of the required number of wells, taking into account the required mixing with 4X LDS loading buffer and 10X Reducing Agent (RA). 10 μg sample per well in 20 μl (for midi gels) is a good starting point for most applications, but this may require adjusting on a case-by-case basis.  ABCDEFG1SamplesProtein concentration
  mg/ml (equivalent to μg/μl)V for loading of
  4 gels with 5 μg per well (prepare for 20 μg in 80 μl, load 20 μl per well)4X LDS10X RALB_top_upFinal V&nbsp;2G7_251.54901960812.920839.1803G7_261.27634803915.720836.3804G7_270.58088235334.420817.6805G7_281.2175245116.420835.6806G7_291.86213235310.720841.3807G7_301.14460784317.520834.5808G7_310.38725490251.62080.4809G7_321.58578431412.620839.48010G7_331.44117647113.920838.180
ABCDEFG1SamplesProtein concentration
  mg/ml (equivalent to μg/μl)V for loading of
  4 gels with 5 μg per well (prepare for 20 μg in 80 μl, load 20 μl per well)4X LDS10X RALB_top_upFinal V&nbsp;2G7_251.54901960812.920839.1803G7_261.27634803915.720836.3804G7_270.58088235334.420817.6805G7_281.2175245116.420835.6806G7_291.86213235310.720841.3807G7_301.14460784317.520834.5808G7_310.38725490251.62080.4809G7_321.58578431412.620839.48010G7_331.44117647113.920838.180
Once the dilutions have been prepared, return the original samples to   for long-term storage. It is also possible to store diluted samples in LDS/RA at  , thaw and heat for loading at a later time.
?. [Cell lysis and protein extraction]
Heat samples at   for   (if prepared in PCR strips, use thermocycler). It is better to use a lower temperature to limit potential protein degradation while still achieving denaturation. Once completed, leave the samples to cool off at  ; pulse-spin to pull-down any condensed liquid.
70 °C
0 Room temperature
?. [Cell lysis and protein extraction]
In the meantime, prepare running buffer according to manufacturers conditions (use 20X MOPS or 20X MES stock buffers - choice will depend on the size of your proteins of interest; use deionised H2O for dilution).
For the inner gel chamber, prepare a separate aliquot of running buffer and supplement with NuPAGE Antioxidant (  per   ).
?. [Transfer, blocking and primary antibody incubation]
Prepare blocking buffer:   BSA to 1X TBS/T (Tween-20 at 0.1 %).
?. [Transfer, blocking and primary antibody incubation]
Prepare the required materials for transfer: trays with EZ gel staining reagent, trays with double-deionised H2O for filter paper wetting, tissue paper, scalpel, forceps, transfer packs, iBlot2 machine.
?. [Transfer, blocking and primary antibody incubation]
When the gel has finished running, dissassemble the casette, cut excess gel material off and transfer the gel using the iBlot2 system according to the manufacturer's instructions (Programme P3 for   is a good starting option, but may require changing based on protein of interest). When assembling the gel transfer stack, wet the filter paper in double-deionised H2O and tap dry on some tissue paper before putting on top of the gel; this will reduce water-copped corrosion and the formation of "green" streaks on the membrane).
?. [Transfer, blocking and primary antibody incubation]
Once transferred, cut the membrane if necessary and decide on a marking system to be able to track the identify of the membrane if multiple membranes are being transferred (e.g. corner triangles cut with scalpel; number identifying gel number). Transfer the membrane to a box with blocking solution covering the entire gel. Put to rock on a shaker for 45 minutes to 1 hour.
?. [Transfer, blocking and primary antibody incubation]
Stain the transferred gels in EZ Blue solution for  , then destain using deionised H2O. Both steps should be performed on a shaker. The gels can be imaged (epi-illumination or 700/800 nm fluorescence) several days later if necessary - leave on bench at room temperature once destaining completed.
?. [Transfer, blocking and primary antibody incubation]
Prepare dilutions of primary antibody in blocking buffer according to the manufacturer's instructions.
?. [Transfer, blocking and primary antibody incubation]
Once the membrane has finished blocking, remove the blocking solution (can be reused), and add the primary antibody dilution so that it covers the entire blot surface. Incubate at  with rotation: typically overnight, but can be left for longer if necessary.
4 °C
?. [Secondary antibody incubation and ECL detection]
Decant used primary antibody into original tube (for reuse) and return to the fride (primary antibody should be supplemented with 0.02 % sodium azide for continued fridge storage).
?. [Secondary antibody incubation and ECL detection]
Wash the membrane 5X for 5 minutes each in 1X TBS/T buffer.
?. [Secondary antibody incubation and ECL detection]
Dilute the required secondary antibody in blocking buffer. We typically use 1:10,000 dilutions of either one of the following antibodies: anti-rabbit IgG HRP-linked antibody (CST #7074S), anti-mouse IgG HRP-linked antibody (7076S).
?. [Secondary antibody incubation and ECL detection]
Incubate the membrane in secondary antibody for   at  on a shaker. Make sure that the solution covers the entire surface of the membrane.
0 Room temperature
?. [Secondary antibody incubation and ECL detection]
Decant secondary antibody and store in fridge for one more reuse if necessary.
?. [Secondary antibody incubation and ECL detection]
Wash the membrane 5X for 5 minutes each in 1X TBS/T buffer.
?. [Secondary antibody incubation and ECL detection]
Prepare ECL solution according to the manufacturer's instructions. Use 2 ml ECL mix per 10 cm x 8 cm membrane.
?. [Secondary antibody incubation and ECL detection]
Place the membrane on a flat surface (e.g. glass plate), making sure that excess washing solution has been drained off, and apply the ECL solution. Ensure that the blot is covered evenly.
?. [Secondary antibody incubation and ECL detection]
Incubate for  , then proceed with detection using a chemiluminescence imaging system.
Tip: make sure the imaging settings are written down in a readme.txt file, alongside experimental information (samples, transfer condition, antibody dilutions and lot numbers, blocking conditions)
?. [Secondary antibody incubation and ECL detection]
Once detected, the membrane can be stored in TBS/T in the fridge for up to two weeks if required for repeated incubations.
?. [Cell lysis and protein extraction]
Vortex each sample for  .
?. [Cell lysis and protein extraction]
In the mean time, thaw previously prepared BSA standards (0 - 2 mg/ml).
?. [Cell lysis and protein extraction]
Load each sample using gel loading tips; load   protein ladder at the start and/or end of the gel (or as required). Run the gel at 120V until the dye front has reached the plate bottom (usual run time  ).
10 µl
For good results, it is absolutely essential to mix the samples before loading!
Note that each midi tank can take 4 gels, however, loading of the gels becomes more difficult when they face backwards and may result in imprecision. It is best to stik to 12-well gels or less to minimise sample diffusion which takes place the longer the samples sit without external voltage during loading.