Protein blot (Western)

Traditional ECL image utilizing a chemiluminescent substrate to react with HRP, producing light that exposes the film in a band pattern

A protein blot, also known as the immunoblot or Western blot, is a method of semiquantitative determination of protein expression. Crude cell lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current passing through the gel will then propel the proteins through the gel, with the largest proteins travelling the slowest. This results in the proteins being roughly separated by their mass. The proteins are then transferred from the gel onto a membrane (often nitrocellulose or PVDF), which is then incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can then specifically detect the protein of interest.

Standard western blot procedure

Prior to running the western blotting experiment, consult with the vendor in order to determine if they have optimized the titration for the antibody you just purchased. Most vendors should offer some guideline for how to use the antibody. Value your time and effort first and foremost. If the experiment comes out strange, make the call or email the vendor for assistance. Your time is the most valuable element to the procedure!

Mouse monoclonal western blot

Load at least 50 ug of whole cell lysate or 20 ug nuclear extract prepared from fresh buffers (30 µg of protein ~ 25,000 cells). Resolve proteins on the gel voltage gradient.
Transfer proteins to a nitrocellulose or PVDF membrane and block the membrane in 10 cm square dish with fresh 5% milk TTBS 2 hr room temperature, or overnight at 4C.
Perform 3-5 shake rinses with cold TTBS (until no residual milk is observed).
Incubate the primary antibody 1:200 in fresh 3% milk TTBS for 90 minutes room temperature.
Perform 3 shake rinses, followed by 4X for 5 minutes washes with ambient or cold TTBS.
Incubate the the secondary antibody 1:1000 anti-mouse in fresh 1% milk TTBS for 60 minutes at room temperature.
Perform 3 shake rinses, followed by 4X for 5 minutes washes with ambient or cold TTBS.
Perform ECL using a suitable detection reagent. Wick away all residual chemiluminescent liquid by dabbing membrane edges on paper towel.
Prepare the membranes for ECL by covering with either saran wrap, or a transparency page. There should be no running of ECL liquid.
Optimize protein signal with multiple exposure times.

Goat polyclonal western blot

Load at least 50 ug of whole cell lysate or 20 ug nuclear extract prepared from fresh buffers (30 µg of protein ~ 25,000 cells). Resolve proteins on the gel voltage gradient.
Transfer proteins to a nitrocellulose or PVDF membrane and block the membrane in 10 cm square dish with fresh 5% milk TTBS 2 hr room temperature, or overnight at 4C.
Perform 3-5 shake rinses (until very little residual milk washes off the membrane).
Incubate membrane with the primary antibody 1:200 in fresh 5% Milk TTBS for 90 minutes room temperature.
Perform 3 shake rinses, followed by 4X 5 minute wash with ambient or cold TTBS.
Incubate the the secondary antibody 1:5000 anti-goat in fresh 5% Milk TTBS for 60 minutes at room temperature.
Perform shake rinses, until no residual milk is observed, followed by 4X 5 minute wash with ambient or cold TTBS.
Perform ECL using a suitable detection reagent. Wick away all residual chemiluminescent liquid by dabbing membrane edges on paper towel.
Prepare the membranes for ECL by covering with either saran wrap, or a transparency page. There should be no running of ECL liquid.
Optimize protein signal with multiple exposure times.

Rabbit polyclonal western blot

Load at least 50 ug of whole cell lysate or 20 ug nuclear extract prepared from fresh buffers (30 µg of protein ~ 25,000 cells). Resolve proteins on the gel voltage gradient.
Transfer proteins to a nitrocellulose or PVDF membrane and block in 10 cm square dish with fresh 5% milk TTBS 2 hr, r.t. or overnight at 4C.
Perform 3 shake rinses (until very little residual milk washes off the membrane).
Incubate the primary antibody 1:200 in fresh 3% milk TTBS for 90 minutes room temperature.
Perform 3 shake rinses, followed by 4X 5 minute wash with ambient or cold TTBS.
Incubate the the secondary antibody 1:4000 anti-rabbit in fresh 1% Milk TTBS for 60 minutes at room temperature.
Perform 3 shake rinses, followed by 4X 5 minute wash with ambient or cold TTBS.
Perform ECL using a suitable detection reagent. Wick away all residual chemiluminescent liquid by dabbing membrane edges on paper towel.
Prepare the membranes for ECL by covering with either saran wrap, or a transparency page. There should be no running of liquid.
Optimize protein signal with multiple exposure times.

Phosphorylation state specific western blot

Adjusting certain incubation conditions can improve the detection signal for the phospho-specific antibody. When serine/threonine phosphatase inhibitor Sodium Fluoride (NaF), and tyrosine phosphatase inhibitor Sodium Orthovanadate (Na3VO4) are included in the blocking and incubation buffers, phospho-specific signals can noticeably improve. Including 50 mM NaF and 5 mM Na3VO4 in the blocking and incubation buffers can improve the signal. Using 5% milk diluent for primary and secondary incubations will reduce the nonspecific banding and background.

Load 50 ug of whole cell lysate/tissue extract OR 20 ug of nuclear extract prepared from fresh buffers. Resolve proteins on the gel voltage gradient.
Transfer proteins to a nitrocellulose or PVDF membrane and block in fresh 5% milk TTBS, 50 mM NaF/5 mM Na3VO4 for 2 hr, r.t. or overnight at 4C. Perform 3 shake rinses and drain until no residual milk appears to stream off the wash buffer.
Incubate the primary antibody 1:200-20000 in fresh 5% milk TTBS, 50 mM NaF/5 mM Na3VO4 for 120 minutes room temperature. Perform 3 shake rinses followed by wash with cold TTBS 4X for 5 minutes each wash.
Incubate the the secondary antibody 1:2000 anti-mouse, 1:5000 anti-rabbit, 1:5000 anti-goat in fresh 5% milk TTBS, 50 mM NaF/5 mM Na3VO4 for 60 minutes at room temperature. Perform 3 shake rinses followed by wash with cold TTBS 4X for 5 minutes each wash.
Perform ECL using a suitable chemiluminescent reagent
Optimize protein signal with multiple exposure times.

Dephosphorylation of Lysate with Lambda Phosphatase

Lysate Treatment

To a micro-centrifuge tube add 80 µg of lysate. Amount maybe scaled up or down as needed.
Add 6 microliters of lambda-PPase Buffer. Final concentration: 50 mM Tris, pH 7.5, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 2 mM MnCl2.
Add 400 units of l-Phosphatase

Santa Cruz Biotechnology Inc. sc-200312

Millipore Cat. #14-405

Incubate at 30˚C for 20 minutes.
The protein sample/lysate is now ready for analysis or further preparation depending on application

Sharma SK and Carew TJ. Inclusion of phosphatase inhibitors during Western blotting enhances signal detection with phospho-specific antibodies. Anal Biochem. 2002 Aug 1;307(1):187-9. DOI:10.1016/s0003-2697(02)00008-8 | PubMed ID:12137799 | HubMed [Paper1]

Membrane Treatment

Treats ~30 lanes or 2-3 (6x12 cm) membranes. Mark treated and untreated membranes.

Block membranes in 3%BSA, 1X TBS, 0.1% Triton X-100.
Add 15 ul of Lambda-PPase (Santa Cruz Biotechnology Inc. sc-200312) in 15 ml of blocking buffer + 2mM DTT, 2mM MnCl2.
Incubate submerged membrane RT, 4hr or 4C ON.
Wash 2x 1X PBST.
Wash 2x dH2O.
1ary incubation / 2ary incubation / wash / ECL.

Heat dependent antigens

Certain antigens (caveolin-1) have been reported to be heat sensitive in SDS-PAGE analysis. Antigen sensitivity may be improved by adjusting lysis heating parameters from

95C for 3-5 minutes

TO

70C for 10 minutes

Transfer tips for high molecular weight proteins

Standard protein transfer

1) Perform electrophoresis on 8-15% acrylamide SDS-PAGE gel, according to standard procedures.

2) Transfer proteins onto a nitrocellulose membrane, overnight (approximately 18 hours) at 0.1 Amp.

3) Running Buffer: 50 mM Tris: 6.06 g Tris Base, 380 mM Glycine: 28.5 g Glycine, 1.6 mM EDTA: 0.67 g EDTA (tetra Na is the most soluble), 0.1% SDS: 1.0 g SDS

4) Transfer buffer: 50 mM Tris, 380 mM glycine, 0.1% (w/v) SDS, 10% (v/v) methanol. Final pH: 8.3.

Adjustments to consider for high molecular weight proteins

6% acrylamide gel is preferable with high molecular weight proteins.
Use 15-20% methanol in transfer buffer. Methanol strips the SDS off of the proteins, which changes the charge of the proteins. Methanol also causes the gel to shrink.
Do not let the gel sit in transfer buffer for prolonged periods prior to transfer.
Transfer for 16 hours at 30 Volts, then 1 hour at 100 Volts. Do not exceed 220 mAmps.
O.45 um PVDF or nitrocellulose may be effective.

Transfer tips for low molecular weight proteins

Tricine Gels are ideal for protein separation from 2.5-50 kDa.

Tricine Gel
Gradient 10-20%
0.1 or 0.05um PVDF or 2 pieces of 0.2 micron PVDF
lower transfer time relative to standard transfer time

Tricine Gel

Tricine ions migrate ahead of the smallest proteins. This improves the resolution of low molecular weight proteins. Tricine gels display good transfer efficiency, in a lower pH.

Tricine Gel Recipe

Protein Deglcyosylation

N-link carbohydrate chains (light blue) influence the actual surface contour of membrane bound proteins (green/red) that harbor them

Glycosylation of proteins and lipids is a post-translational modification important for numerous physiological processes; protein folding, ligand binding, cell surface contour, and influencing cell–cell and cell–matrix interactions.

In order to determine the presence and/or significance of protein glycosylation, the carbohydrate chains may be removed by catalysis.

N-Glycosidase F (PNGase F) is a ~36 kDa amidase that cleaves at the innermost N-acetylglucosamine (GlcNAc) and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins.

PNGase F
Endo-O-Glycosidase
a-2(3,6,8,9)-Neuraminidase (Sialidase A)
ß(1,4)-Galactosidase
ß-N-Acetylglycosaminidase

(1) Tarentino, A.L. and Plummer, T.H., Jr. (1994) Methods in Enzymology, 230: 44-47. PMID: 8139511

Deglycosylation kits

Enzymes and reagents combined into convenient to use kits exist in order to remove N-linked and O-linked carbohydrates from glycoproteins, and cleave complex core O-linked carbohydrates (including polylactosamine). Deglycosylation can simplify SDS-PAGE by decreasing the potential broad size variation associated with glycoproteins.

New England Biolabs offers a convenient kit Cat#P0704S.

Sigma Aldrich offers the PNGase F enzyme Cat#P7367.

Troubleshooting

Western Blot Optimization

Blocking: To block nonspecific sites on the membrane, incubate the membrane with blocking buffer (5% milk TTBS) prior to primary antibody incubation. 2 hour room temperature blocking is sufficient for the membrane to absorb proteins and minimize noise.

Primary antibody: Use a diluent containing a blocking protein (5% milk TTBS or 1%milk/1%BSA TTBS) to dilute the primary antibody.

Secondary antibody: Use a diluent containing a blocking protein (5% milk TTBS or 1%milk/1%BSA TTBS) to dilute the primary antibody. Performing a "Secondary control" control blot where the primary antibody is purposely omitted is useful for identifying whether nonspecific signals are due to the secondary antibody conjugate or the primary antibody.

Washes: All wash steps are critical for reducing general background signal and nonspecific binding to discrete bands. If high background is a problem, the number, length and composition of the washes can all be increased.

General: Handle membranes carefully with forceps to minimize problems with nonspecific signals. Wear gloves for all steps to prevent hand contact with film, membranes or detection reagents.

PVDF versus Nitrocellulose

The western blotting procedures are the same for PVDF or nitrocellulose, however the handling of these membranes are different prior to- and during- transfer of proteins from the SDS-PAGE gel to the membrane.

Nitrocellulose

Nitrocellulose exhibits the highest sensitivity with very low backgrounds in all transfers, especially in protein blotting. Unlike PVDF, nitrocellulose wets out naturally, does not require methanol, and will not turn hydrophobic during the transfer process. Nitrocellulose is very easily blocked and does not need the many blocking steps required with PVDF. Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions.

PVDF

Polyvinylidene Fluoride is hydrophobic and should be prewet in methanol before it is used.

Wet the membrane in 100% methanol for 15 seconds. The membrane should uniformly change from opaque to semi-transparent.
Place the semi-transparent membrane into ultrapure water and soak for 5 minutes.
Place the membrane into transfer buffer and soak for at least 5 minutes.
Transfer proteins to the smooth/reflective side that has more shine

Another change to note is that the SDS tolerances are not equivalent for PVDF and Nitrocellulose. The binding of protein to PVDF is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05% for PVDF.

Membrane Stripping

Griffin:Membrane_Stripping_and_Reprobing

Stability of reagents

Ammonium persulphate/persulfate (APS) is a very reactive compound (used for radical generation in the polymerisation process). It is prone to oxidation and loss of activity which will increase your polymerisation times. Make small aliquots, keep in the cold. [1]
TEMED, the catalyst of polymerisation, is also prone to oxidation especially if mixed with water which it attracts if the container is left open. Seal quickly, keep in cold place. [2] [3]

Toxicity of reagents

acrylamide/bis-acrylmide is a neurotoxin. Handle with precaution. [4]
TEMED is very corrosive. Most likely exposure in the lab due to inhalation. Reseal the container as quickly as possible. [5]

Curators

User:Korey Griffin