Staining Western Blots

STAINING WESTERN BLOTS

Total Protein Staining and Immunostaining are procedures applicable to proteins bound to nitrocellulose membranes. The proteins may be applied to a nitrocellulose membrane by electrophoretic transfer, dot or slot blotting, tissue printing, etc. Usually the nitrocellulose is stained first for total protein and photographed (Total Protein Staining ). The stain is then removed and the nitrocellulose membrane restained immunologically, using antibodies specific of the protein of interest (Immunostaining).

Total Protein Staining

A. Reagents and Solutions

CPTS Staining Solution - (0.5 mg/mL CPTS) Dissolve 50 mg of 3,4',4 ²,4-copper phthalocyanine tetrasulfonic acid, tetrasodium salt (CPTS) in 100 mL of 12 mM HCl. The solution is stable at room temperature indefinitely.
Wash Solution - (12 mM HCl, pH 2) Dilute 1.0 mL conc. HCl in 999 mL dH₂O. This solution is stable forever at room temperature.
Optional: Destain Solution - (250 mM NaHCO₃, pH 9.0) Dissolve 2.1 g NaHCO₃ in 100 mL dH₂O and if necessary, adjust the pH to 9.0 with NaOH.

B. Protein Staining Procedure (Wear gloves!)

During each incubation step throughout the procedure, turn over the membrane at least once.
Wash - Wet the nitrocellulose membrane by laying it on top of ~20 mL wash solution.
Staining - Decant the wash solution and add ~10 mL CPTS stain solution. Incubate for 1 minute with gentle agitation, or until staining is complete.
Wash - Decant the stain solution and wash 3X in wash solution with gentle agitation to remove unbound stain.
Photography - the stained nitrocellulose can be photographed on a light box, using Polaroid Type 667 film at f16 and 1/125 sec. with an orange filter.
Mark the position of the protein bands with pencil or India Ink before destaining or immunostaining.
Because we will proceed right away with the immunostaining, no special treatment is required for destaining the membrane: the blocking solution (see immunostaining bellow), saturated with BSA, will compete with the dye and naturally destain the membrane as the blocking occurs
Optional destain - Add 20 mL of destain solution and incubate with gentle agitation, until destaining is complete. Proceed to antibody staining or dry as above

Immunostaining

Immunostaining is achieved with 2 antibodies: the primary antibody, raised in rabbit, is specific for human salivary amylase, and the secondary anibody, raised in goats, is specific for rabbit immunoglobulins. In addition, the secondary antibody is coupled with an enzymatic system able to produce a dark precipitate in presence of its substrate. If the protein of interest is present on the nitrocellulose membrane, it will first be recognized by the primary antibody. When the secondary antibody is applied, it will bind to the primary anitbodies that are already bound to the protein to detect. After extensive washing to remove any unbound antibodies, you can then feed the coupled enzymatic system with its substrate, and the dark precipitate will appear on the blot where the protein to detect is. This system is widely used in biochemistry, in particular because it is highly specific to each protein and allows the detection and staining of very small amounts of protein.

As useful controls when antibody staining, before blocking the membrane, on unused areas of the nitrocellulose apply three separate small drops; one of antigen-containing solution (i.e., a drop of saliva for amylase antibodies), and one each of the primary and secondary antibodies.

A. Reagents and Solutions

10X TBS - (Tris-buffered saline) 250 mM Tris/Tris ×HCl, 1.5 M NaCl, pH 7.5
TBS - (Tris-buffered saline) 25 mM Tris/Tris× HCl, 0.15 M NaCl, pH 7.5
TTBS - TBS with 0.05% (w/v) Tween 20
Blocking solution - 3% (w/v) BSA in TBS
Antibody buffer - 1% (w/v) BSA in TTBS
1st antibody solution - dilute 1st antibody to an appropriate titer (1:10,000 is common, for 1mg/mL) in antibody buffer.
2nd antibody solution - dilute 2nd antibody (conjugated to alkaline phosphatase) (1:20,000 for 0.5 mg/mL) in antibody buffer.
Color development solution -(Prepared from Sigma FAST BCIP/NBT Buffered Substrate Tablets B-5655) 0.1 M Tris buffer, pH 9.5 containing 5 mM MgCl₂, 0.15 mg/mL BCIP (5-bromo-4-chloro-3-indoyl phosphate) and 0.30 mg/mL NBT (nitro blue tetrazolium).

B. Procedure (Wear gloves!)

Throughout the procedure, turn over the membrane at least once during each incubation step.
Apply three separate small drops; one of antigen-containing solution (i.e., a drop of saliva for amylase antibodies), and one each of the primary and secondary antibodies, to the dry membrane..
Blocking - Mark molecular weight standards with pencil or India Ink before blocking. Transfer 10 - 20 mL of blocking solution to a large weighing boat. Lay the membrane on top of the solution and wait for it to absorb the solution. Dry nitrocellulose will darken as it wets. After the membrane is uniformly wet, swirl the solution over the top of the membrane. Incubate for 15 min to 1 hr at room temperature (RT), with gentle agitation.
Wash - decant the blocking solution and add 20 mL TTBS. Wash at RT for 5 - 10 min with gentle agitation.
1st antibody incubation - decant the TTBS and add 10 mL of the 1st antibody solution. Incubate for 30 min to 2 hr at RT, with gentle agitation.
Wash - decant and save the 1st antibody solution. To remove unbound antibody, wash the membrane 2X with 20 mL TTBS for 5 min/wash at RT.
2nd antibody - decant the TTBS and add 10 mL of the 2nd antibody solution. Incubate for 30 min to 2 hr at RT with gentle agitation.
Wash - decant and save the 2nd antibody solution. To remove unbound 2nd antibody, wash the membrane 2X with 20 mL TTBS for 5 min/wash at RT, then, in a new weighing boat rinse with 20 mL TBS for at least 5 min to remove residual Tween 20 from the membrane surface.
Color development - Decant the TBS and add 5 - 10 mL of the color development solution.
Incubate until reddish-brown bands are clearly visible.
Final wash - when the color is satisfactory, decant the color development buffer and wash 2X with 20 mL dH₂O for 5 min/wash .
Photography - the stained nitrocellulose can be photographed on a light box, using Polaroid Type 667 film at f22 and 1/125 sec. (no filter).
Storage - Dry the membrane on filter paper and store between paper or polyester sheets. Do not store in plastic bags, as this may cause the nitrocellulose to decompose. Protect from light.