TITLE: Western Blotting
AUTHORS: Karina Conkrite

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">From growth to isolation and lysis, to quantification, and running the gel, transfer, blotting... the whole bit.</div></div>

[STEPS]
?. [Before Starting:]
Before Starting:Prepare fresh Lysis Buffer for cells. Use 5X stock stored at 4C. Stock solution consists of   ABC1Component1X5X2Tris25 mM125 mM3NaCl150 mM750 mM4EGTA1 mM5 mM5EDTA1 mM5 mM6NaF10 mM50 mMRemaining ingredients to be added fresh to each sample:Final concentration1 mM DTT1% Triton X-1001X Protease/Phosphatase Inhibitor (Cell Signalling, Cat #5872)Per 1 mL 1X buffer:200 uL 5X stock100 uL 10% Triton X-10010 uL 0.1M DTT10 uL 100X Protease/Phosphatase Inhibitor680 uL ddH2OLysis buffer must be made fresh daily and stored during use on ice.
ABC1Component1X5X2Tris25 mM125 mM3NaCl150 mM750 mM4EGTA1 mM5 mM5EDTA1 mM5 mM6NaF10 mM50 mM
?. [Before Starting:]
Grow Cells in culture and treat with whatever desired checmicals/drugs/siRNA/plasmid/etc are desired. Generally, protein is collected 72 hours post treatment. Occasionally, the proteins being monitored may show optimal change at 48 or 96 hours, dependent on cell cycle and protein cycling.
?. [Before Starting:]
Remove media and wash cells with DPBS.
?. [Collecting Cells]
Collect Cells and either stored as a pellet for lysis later, or lyse directly now,
?. [Collecting Cells]
Lyse cells on ice for 15-30 minutes.
?. [Collecting Cells]
Sonicate with probe sonicator (Brodeur Lab, setting 6) 5 seconds to break membranes
?. [Collecting Cells]
Spin down lysis, 4C, 15 min, 15000g or more, 15'.
4 °C
?. [Collecting Cells]
Transfer supernatant to clean tube, without the pellet. This may be stored at -80C. Avoid freeze/thaw cycles. Aliquot if necessary.
?. [Quantify protein]
Run a BCA assay, using the Pierce BCA assay kitEnsure all samples are within the standard curve.
?. [Collecting Cells]
Lyse cells on ice, 15-30 min
?. [Collecting Cells]
Sonicate samples, (Probe sonicator, Brodeur Lab, setting 6) 5 seconds
?. [Collecting Cells]
Spin samples, 4C, 15', at least 15000g.
4 °C
?. [Collecting Cells]
Without disturbing the pellet, collect supernatant and transfer to clean labeled tube.Can be stored at -80C. Avoid freeze/thaw cycles. Aliquot if necessary.
?. [Quantification]
Must quantify protein levels to know how much to load on a gel.Run Pierce BCA assay
?. [Quantification]
Ensure your samples are somewhere on the curve. If they are outside the curve, you will need to redo the assay with different dilution of sample:lysis buffer.
?. [Prep the samples for running]
Select the type and percentage gel you wish to run. We tend to use Tris-Glycine gels. Percentage depends on your proteins of interest.
?. [Prep the samples for running]
Decide how much protein is necessary to run/sample (ug), 15ug? 30 ug?Still keeping samples on ice, aliquot that amount, plus a suitable amount of 2X sample buffer (stored at -20C) and lysis buffer to make up to a standard volume for all the samples. Keep all samples on ice,  until the gel is ready to run.Don't forget to prep your ladders at this time as well.2X SDS Gel Loading Buffer/ Laemmli Buffer  ABCD1ComponentEnd
  Concentrationvol for 4 mLvol for 40 mL230.5 M Tris-HCl pH 6.8125 mM1 mL104Glycerol20%0.8 mL8520% SDS5%0.8 mL86b-Mercaptoethanol10%0.4 mL470.5% Bromophenol Blue0.025%0.2 mL28H2O0.8 mL8
0 °C
ABCD1ComponentEnd
  Concentrationvol for 4 mLvol for 40 mL230.5 M Tris-HCl pH 6.8125 mM1 mL104Glycerol20%0.8 mL8520% SDS5%0.8 mL86b-Mercaptoethanol10%0.4 mL470.5% Bromophenol Blue0.025%0.2 mL28H2O0.8 mL8
?. [setting up the gel]
Dependent on the gel type and transfer type you are doing, prep necessary buffers.For example, if you are running 2 Tris-Glycine gels (Criterion or Novex, both take the same amount of buffer) you will use about 1L TG running buffer.
?. [setting up the gel]
To make 1L of 1X TG Running buffer:  ABC1ComponentVolumeend conc210X Tris-Glycine buffer, BioRad 1610771100 mL1X320% SDS, Technova S02935 mL1%4H2O895mLThis buffer may be stored at  for a time. If using the Criterion set up, buffer may be reused in the tank, but should be fresh in the loading chamber.If using the SureLock boxes, you will use the entire liter every time.Also, prep for the transfer. This is very dependent on the size of your proteins of interest. If you are interested in proteins over 150 kDa, you will want to perform a fully wet transfer, possibly overnight in the cold room. In that case, for TG gels, you need to prep the buffer for the transfer, cut the membrane (1) and 3M filter papers (4) necessary to sandwich the transfer.
ABC1ComponentVolumeend conc210X Tris-Glycine buffer, BioRad 1610771100 mL1X320% SDS, Technova S02935 mL1%4H2O895mL
4 °C
?. [setting up the gel]
Remove plastic barriers on the bottom of pre-cast gels. If you forget to do this, your proteins will not migrate.Assemble the gel/box cartridge, locking everything in place. Higher gel casing should be to the outside of the gel box, so you will load from behind.  Fill buffer chambers with running buffer and remove combs from gels carefully, so as not to disturb the well edges and displace them.Using running buffer and a p200 or p1000 pipet, flush the wells to remove the storage buffer.
?. [setting up the gel]
While you are flushing the wells, heat samples according to the gel type. For example, on a TG gel, samples should be boiled at   WITH LID LOCKS!!! for Check the directions for the ladder you have selected. Different ladders have different instructions. Some need no boiling at all, some are 2 min, adjust accordingly.
95 °C
[use lid locks]
?. [setting up the gel]
Briefly spin down the tubes to collect samples. Load into gel according to your map using gel loading tips or Rainin p20 tips.
?. [Run the gel]
Run the gel according to gel type. For TG gels, we prefer to start slowly (75V) to get through the stacking gel (~20 min). Voltage can then be increased to run faster according to gel type/percentage. Run samples to the bottom of the gel. You can find the optimal voltage/amperage rates for your gels on the info sheets from their supplier.
?. [Set up the transfer]
While gels are running, prep your transfer method.This will depend on your proteins of interest. Low to medium molecular weight proteins (20-150 kDa) can take advantage of the TransBlot Turbo system in the lab. If you are interested in higher molecular weight proteins, a more traditional wet transfer is called for. Recommended is overnight transfer in the cold room.IN ALL CASES, prep your necessary buffer ahead of time so it is cold.
?. [TRANSFERRING THE GEL TO THE MEMBRANE]
Once you are set up with the appropriate sandwich/buffer/apparatus combo....Check your current! And transfer...
?. [Check the transfer]
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.
?. [Blotting]
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking. For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.This will help cut down on non-specific binding of antibodies.Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and  set on stir plate to mix until uniform.
?. [Blotting]
If splitting membrane into sections, cut now. This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.
?. [Blotting]
Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with  3.2 uL beta-Actin.Incubate overnight at
[while rocking]
?. [Blotting]
Remove blot from 4C, rinse quickly in TBST, then wash 3 X 10' in TBST, with rocking.During the last wash, prepare the secondary antibody
?. [Blotting]
Secondary antibody:whatever the primary antibody's host species was, you need an antibody against that.We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as at a 1:25000 dilution in 5% milk in TBST.Apply and incubate with rocking, 1 hour
?. [Blotting]
Discard the secondary antibody, rinse blot quickly in TBST, then wash 3 X 10' in TBST, with rocking.
?. [Blotting]
Develop the blot, using the ECL detection method of choice. Lowly expressed proteins, recommend for other, more abundant proteins, less sensitive substrates may be used. carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST. Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
?. [Blotting]
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
?. [Stripping]
if you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL and incubate in the hyb oven at   while turning, for 15-20 min
37 °C
?. [Stripping]
Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
?. [Stripping]
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions.  Follow as before, as many times as desired.
?. [Check the transfer]
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.
?. [Check the transfer]
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.
?. [Blotting]
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking. For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.This will help cut down on non-specific binding of antibodies.Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and  set on stir plate to mix until uniform.
?. [Blotting]
If splitting membrane into sections, cut now. This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.
?. [Blotting]
Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with  3.2 uL beta-Actin.Incubate overnight at
[while rocking]
?. [Blotting]
Remove blot from 4C, rinse quickly in TBST, then wash 3 X 10' in TBST, with rocking.During the last wash, prepare the secondary antibody
?. [Blotting]
Secondary antibody:whatever the primary antibody's host species was, you need an antibody against that.We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as at a 1:25000 dilution in 5% milk in TBST.Apply and incubate with rocking, 1 hour
?. [Blotting]
Discard the secondary antibody, rinse blot quickly in TBST, then wash 3 X 10' in TBST, with rocking.
?. [Blotting]
Develop the blot, using the ECL detection method of choice. Lowly expressed proteins, recommend for other, more abundant proteins, less sensitive substrates may be used. carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST. Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
?. [Blotting]
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
?. [Stripping/Reprobe]
if you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL and incubate in the hyb oven at   while turning, for 15-20 min
37 °C
?. [Stripping/Reprobe]
Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
?. [Stripping/Reprobe]
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions.  Follow as before, as many times as desired.
?. [Check the transfer]
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking. For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.This will help cut down on non-specific binding of antibodies.Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and  set on stir plate to mix until uniform.
?. [Check the transfer]
If splitting membrane into sections, cut now. This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.
?. [Check the transfer]
Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with  3.2 uL beta-Actin.Incubate overnight at
[while rocking]
?. [Check the transfer]
Remove blot from 4C, rinse quickly in TBST, then wash 3 X 10' in TBST, with rocking.During the last wash, prepare the secondary antibody
?. [Check the transfer]
Secondary antibody:whatever the primary antibody's host species was, you need an antibody against that.We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as at a 1:25000 dilution in 5% milk in TBST.Apply and incubate with rocking, 1 hour
?. [Check the transfer]
Discard the secondary antibody, rinse blot quickly in TBST, then wash 3 X 10' in TBST, with rocking.
?. [Check the transfer]
Develop the blot, using the ECL detection method of choice. Lowly expressed proteins, recommend for other, more abundant proteins, less sensitive substrates may be used. carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST. Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
?. [Check the transfer]
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
?. [Check the transfer]
if you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL and incubate in the hyb oven at   while turning, for 15-20 min
37 °C
?. [Check the transfer]
Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
?. [Check the transfer]
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions.  Follow as before, as many times as desired.
?. [TRANSFERRING THE GEL TO THE MEMBRANE]
Once you are set up with the appropriate sandwich/buffer/apparatus combo....Check your current! And transfer...
?. [Check the transfer efficiency]
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.
?. [Blotting]
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking. For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.This will help cut down on non-specific binding of antibodies.Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and  set on stir plate to mix until uniform.
?. [Blotting]
If splitting membrane into sections, cut now. This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.
?. [Blotting]
Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with  3.2 uL beta-Actin.Incubate overnight at
[while rocking]
?. [Blotting]
Remove blot from 4C, rinse quickly in TBST, then wash 3 X 10' in TBST, with rocking.During the last wash, prepare the secondary antibody
?. [Blotting]
Secondary antibody:whatever the primary antibody's host species was, you need an antibody against that.We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as at a 1:25000 dilution in 5% milk in TBST.Apply and incubate with rocking, 1 hour
?. [Blotting]
Discard the secondary antibody, rinse blot quickly in TBST, then wash 3 X 10' in TBST, with rocking.
?. [Blotting]
Develop the blot, using the ECL detection method of choice. Lowly expressed proteins, recommend for other, more abundant proteins, less sensitive substrates may be used. carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST. Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
?. [Blotting]
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
?. [Stripping/ Reprobe]
if you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL and incubate in the hyb oven at   while turning, for 15-20 min
37 °C
?. [Stripping/ Reprobe]
Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
?. [Stripping/ Reprobe]
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions.  Follow as before, as many times as desired.
?. [TRANSFERRING THE GEL TO THE MEMBRANE]
Once you are set up with the appropriate sandwich/buffer/apparatus combo....Check your current! And transfer...
?. [Check the transfer efficiency]
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.
?. [Blotting]
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking. For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.This will help cut down on non-specific binding of antibodies.Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and  set on stir plate to mix until uniform.
?. [Blotting]
If splitting membrane into sections, cut now. This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.
?. [Blotting]
Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with  3.2 uL beta-Actin.Incubate overnight at
[while rocking]
?. [Blotting]
Remove blot from 4C, rinse quickly in TBST, then wash 3 X 10' in TBST, with rocking.During the last wash, prepare the secondary antibody
?. [Blotting]
Secondary antibody:whatever the primary antibody's host species was, you need an antibody against that.We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as at a 1:25000 dilution in 5% milk in TBST.Apply and incubate with rocking, 1 hour
?. [Blotting]
Discard the secondary antibody, rinse blot quickly in TBST, then wash 3 X 10' in TBST, with rocking.
?. [Blotting]
Develop the blot, using the ECL detection method of choice. Lowly expressed proteins, recommend for other, more abundant proteins, less sensitive substrates may be used. carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST. Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
?. [Blotting]
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
?. [Stripping/Reprobe]
If you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL and incubate in the hyb oven at   while turning, for 15-20 min
37 °C
?. [Stripping/Reprobe]
Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
?. [Stripping/Reprobe]
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions.  Follow as before, as many times as desired.
?. [Check transfer efficiency]
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.
?. [Blotting]
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking. For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.This will help cut down on non-specific binding of antibodies.Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and  set on stir plate to mix until uniform.
?. [Blotting]
If splitting membrane into sections, cut now. This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.
?. [Blotting]
Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with  3.2 uL beta-Actin.Incubate overnight at
[while rocking]
?. [Blotting]
Remove blot from 4C, rinse quickly in TBST, then wash 3 X 10' in TBST, with rocking.During the last wash, prepare the secondary antibody
?. [Blotting]
Secondary antibody:whatever the primary antibody's host species was, you need an antibody against that.We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as at a 1:25000 dilution in 5% milk in TBST.Apply and incubate with rocking, 1 hour
?. [Blotting]
Discard the secondary antibody, rinse blot quickly in TBST, then wash 3 X 10' in TBST, with rocking.
?. [Blotting]
Develop the blot, using the ECL detection method of choice. Lowly expressed proteins, recommend for other, more abundant proteins, less sensitive substrates may be used. carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST. Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
?. [Blotting]
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
?. [Stripping/Reprobe]
if you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL and incubate in the hyb oven at   while turning, for 15-20 min
37 °C
?. [Stripping/Reprobe]
Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
?. [Stripping/Reprobe]
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions.  Follow as before, as many times as desired.