Protein blot (Western)
Curators
Abstract
The Western blot is one of the most tried and true experiments for antibody-dependent determination of protein expression/presence in a sample.
Materials
PVDF or Nitrocellulose membrane: 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 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 is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes. 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.
Reagents
Primary antibody
Secondary antibody
Detection reagent
Positive control sample
Equipment
- Gel Apparatus
- Pipet
- 10 cm square trays
- PVDF or Nitrocellulose
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!
I) 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.
II) Transfer proteins to a nitrocellulose or PVDF membrane and block in fresh 5% milk TTBS 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.
III) Incubate the primary antibody 1:200-20000 in fresh 5% milk TTBS for 120 minutes room temperature. Perform 3 shake rinses followed by wash with cold TTBS 4X for 5 minutes each wash.
IV) Incubate the the secondary antibody 1:2000 anti-mouse, 1:5000 anti-rabbit, 1:5000 anti-goat in fresh 5%milk TTBS for 60 minutes at room temperature. Perform 3 shake rinses followed by wash with cold TTBS 4X for 5 minutes each wash.
V) Perform ECL using a suitable chemiluminescent reagent
VI) Optimize protein signal with multiple exposure times.
PHOSPHORYLATION SPECIFIC IMMUNOBLOT PROTOCOL
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.
I) 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.
II) 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.
III) 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.
IV) 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.
V) Perform ECL using a suitable chemiluminescent reagent
VI) Optimize protein signal with multiple exposure times.
- 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 |
Critical steps
Primary antibody incubation time:
Primary antibody incubation buffer:
Secondary antibody incubation buffer:
Incubation buffers: If the primary and secondary antibody incubation buffers contain BSA or no blocking protein and the result is a high background to almost a completely black blot, then the addition of 5% milk to both the primary and secondary antibody dilution buffers should improve the result. Try Blocking/Primary/Secondary incubations all in 5% milk TTBS.
Troubleshooting
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.
Notes
Transfer buffer formulation:
- 25 mM Tris-Base
- 192 mM Glycine
- 20% Methanol
- pH 8.3
For 1 L of buffer mix 3.03 g of Tris-Base, 14.4 g of glycin and 200 mL of methanol
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