Immunoblot validation of phospho-specific antibodies using lung cancer cell lines

Abstract

The cancer phenotype is usually characterized by de-regulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both pre-clinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and un-phosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in de-phosphorylating the target protein and assessing if such de-phosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines. The protocol consists in the de-phosphorylation of the Rb-containing protein lysates by treating them with bovine intestinal phosphatase, followed by assessment of the de-phosphorylation by immunoblot.

1. Introduction

The cancer phenotype is usually characterized by overactivation of signal transduction pathways that include components with kinase activity. As a consequence of the overactivation of such pathways, abnormally high levels of phosphorylation of several intracellular proteins occurs, and this hyper-phosphorylation usually has a disruptive effect on the normal regulation of protein function, which is in turn conducive to the cancer state. For example, the MAPK pathway can be abnormally over-active in cancer cells, an over-activation that can arise as a consequence of several mutational events, such as amplification or activation of receptor tyrosine kinases or gain-of-function mutations in the Ras protein [1–3]. Over-activation of the MAPK pathway in the context of cancer or of rapidly diving cells, results in phosphorylation and activation of MAPK intermediaries such as Erk as part of an aberrant cancer-associated signaling that leads to uncontrolled cell proliferation [4]. As another example, the retinoblastoma (Rb) protein, one of the most important cellular tumor suppressors, is inactivated by hyper-phosphorylation in rapidly dividing cells, including cancer cells [5–8]. These, and many other examples of proteins whose function is regulated by phosphorylation has created a need to develop phospho-specific antibodies to specifically detect the phosphorylated variants of proteins whose function is regulated at the phosphorylation level. Expectedly, these phospho-specific antibodies have become important tools for molecular studies of cancer cells and tissues, especially when hyper-phosphorylation of key proteins is associated with the cancer phenotype, as well as in the clinic in the context of pathological assessment of tumors.

Phospho-specific antibodies can be used to detect phosphorylated proteins in tissue sections by immunohistochemistry and by immunoblotting of protein lysates from cancer cell lines and cancer tissue biopsies. Obtaining reproducible results with these antibodies relies in great measure on their preliminary validation, including ensuring that they are indeed able to discriminate between the phosphorylated and un-phosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. Determining this can be relatively straightforward, and the approach consists in de-phosphorylating the target protein and assessing if such de-phosphorylation abrogates immunoreactivity in either an immunoblot or in an immunohistochemistry assay, or if it generates an electrophoretic mobility shift consistent with de-phosphorylation as visualized in an immunoblot.

In this chapter, we describe a protocol to de-phosphorylate protein lysates obtained from lung cancer cell lines by treating them with a phosphatase enzyme, specifically with bovine intestinal phosphatase. This is followed by subjecting such de-phosphorylated lysates to immunoblot analyses using a phospho-specific antibody, in our case against Rb phosphorylated in Serine 249 (S249). We have recently identified this phosphorylation as a biomarker predicting poor prognosis in lung cancer [9]. In an immunoblot assay, successful de-phosphorylation should be observed in the form of an altered electrophoretic mobility of the protein of interest, since removal of phosphate groups affect a protein’s molecular weight, or as a complete abrogation of immunoreactivity when using the phospho-specific antibody in either immunoblots or immunohistochemistry.

2. Materials

All the solutions were prepared using distilled water, unless otherwise stated. Reagents and solutions were stored at room temperature, unless specified. Follow all waste disposal regulations when disposing waste materials.

2.1. Cell Lysis and Protein Extraction and Quantification

1. Cell lines of your choice. The procedure described below requires that the user has cell cultures ready for protein extraction. We optimized the procedure using a variety of lung cancer cell lines to detect total and phosphorylated Rb in them, but this protocol is adaptable to any cell line that expresses the phospho-protein of interest. It is important that you grow your cell cultures in the appropriate medium such that they are at approximately 90–95% confluence at the moment of protein extraction.

2. 1X Phosphate-Buffered Saline (PBS), pH 7.2. To prepare, dissolve 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄^.2 H₂O and 0.24 g of KH₂PO₄, in 800 mL of water. Adjust the pH to 7.2 with HCl and add distilled water to complete the volume to 1 L. Pre-made, ready-to-use PBS can also be purchased.

3. Trypsin-EDTA solution. This is for detaching cells from culture plates. We purchase this solution in pre-mixed, read-to-use form. Alternatively, cells can also be mechanically detached using a rubber scraper.

4. RIPA lysis buffer. For convenience, we use a commercially available pre-mixed 10X RIPA buffer, which is diluted to 1X when using. This solution can also be prepared in the laboratory using the standard recipe, which consists of 10 mM Tris-HCL, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% (v/v) NP-40 (or 1% Triton X-100, if NP-40 is not available), 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) SDS, and 150 mM NaCl.

5. Broad specificity protease inhibitor cocktail, use according to manufactureŕs specifications (see Note 1).

6. Protein concentration determination assay reagents. We perform a standard protein quantification using Bradford Assay, following its instructions, and using BSA as a quantification standard. We use Bio-Rad’s Protein Assay Dye Reagent Concentrate, but other substitute assays can be used.

2.2. Alkaline Bovine Intestinal Phosphatase (BIP) Treatment of Protein Lysates

1. Alkaline phosphatase from bovine intestinal mucosa (bovine intestinal phosphatase or BIP). We have optimized this protocol with the one provided by Sigma-Aldrich (Cat. No. P0114–10KU), provided at a specific activity of 6694 DEA Units/mg protein, and at a concentration of 123 Units/μL. Other commercially available phosphatases can be used as long as the manufacturer’s indications are followed.

2. 10X Dephosphorylation Buffer. This is the buffer for the BIP reaction. To prepare, dissolve 6.07 g of Tris base (50 mM), 5.84 g of NaCl (100 mM), 0.97 g of MgCl₂ (10 mM) and 0.16 g of DTT (1mM) in 800 ml distilled water, stir the solution until the reagents are dissolved. Adjust the pH to 7.9 and complete to a final volume of 1L with distilled water. Numbers in parentheses are the molar concentrations of each component in the final solution. Use Table 1 as a reference for the components and their final concentrations in the working solution.

3. 2X BIP inhibitor cocktail. To prepare, dissolve 0.042 g of sodium fluoride (NaF), 0.092 g of sodium orthovanadate (Na₃VO₄) and 0.013 g of sodium pyrophosphate decahydrate 99% (NaPP) in 4 mL of distilled water in a 15 mL conical tube. Vortex the contents to ensure complete dissolution of the components, complete volume with distilled H₂O to 5 mL. Use Table 2 as a reference for the components and their final concentrations in the working solution. This should be a 2X solution with concentrations of 50 mM Na₃VO₄, 5 mM NaPP and 100 mM NaF that, when brought down to 1X in the final reaction mixture should be enough to inhibit the activity of 100 Units of BIP [10–14].

Table 1.

Amounts and final working concentrations of each of the reagents used for the preparation of the 10X dephosphorylation buffer.

Reagent	Molecular Weight	Amount to weight	Expected final concentration
Tris	121.14 g/mol	6.07 g	50 mmol/L
NaCl	58.44 g/mol	5.84 g	100 mmol/L
Mgcl2	95.21 g/mol	0.97 g	10 mmol/L
DTT	154.25 g/mol	0.16 g	1 mmol/L

Table 2.

Amounts and final working concentrations of each of the reagents used for the preparation of the 2X phosphatase inhibitors solution

Reagent	Molecular Weight	Amount to weight	Expected final concentration
Sodium Fluoride	42.0 g/mol	0.042 g	200 mmol/L
Sodium orthovanadate	265.9 g/mol	0.092 g	100 mmol/L
Sodium Pyrophosphate	183.9 g/mol	0.013 g	10 mmol/L

2.3. SDS Polyacrylamide Gel Electrophoresis

1. 1.5 M Tris–HCl, pH 8.8. Dissolve 181.7 g of Tris base in 800 ml H₂O. Adjust pH to 8.8 with concentrated HCl (use less concentrated HCL as you approach the desired pH). Add H₂O to complete the volume to 1 L. Store at 4 °C.

2. 0.5 M Tris–HCl, pH 6.8. To prepare, dissolve 60.6 g of Tris base in 800 ml H₂O. Adjust pH to 6.8 with concentrated HCl (use less concentrated HCL as you approach the desired pH). Add H₂O to complete the volume to 1 L. Store at 4 °C.

3. 30% acrylamide/Bis-acrylamide solution, can be purchased in ready-to-use form (see Note 2).

4. Ammonium persulfate (APS) 10% (w/v) in water (see Note 3). Prepare by dissolving 0.5 g of ammonium persulfate in 5 ml of H₂O.

5. Tetramethylethylenediamine (TEMED).

6. 10% SDS. Dissolve 10 g of SDS in 80 ml H₂O. Complete volume to 100 ml. This solution can be kept at room temperature for up to 6 months.

7. 2X SDS sample loading buffer: 100 mM Tris-HCl pH 6.8, 4% (w/v) sodium dodecyl sulfate (or SDS, electrophoresis grade), 0.2% bromophenol blue, 20% (v/v) glycerol and 200 mM dithiothreitol (DTT).

8. 10X SDS PAGE running buffer. Dissolve 30.0 g of Tris base, 144.0 g of glycine and 10.0 g of SDS in 1 L of H₂O. Check that the pH is 8.3 but is expected that minimal or no pH adjustment will be required. Store the running buffer at room temperature and dilute to 1X before use.

9. Ethanol 70% (for cleaning electrophoresis glass plates).

10. Protein ladder molecular weight marker. When blotting for Rb, we use Bio-Rad Precision Plus Protein Kaleidoscope (Cat. No. 1610–375). You can use any other protein ladder provided it has sufficient markers in the molecular weight range of your protein of interest, in our case, around 110 kDa, which is the molecular weight of Rb.

2.4. Transfer

1. Nitrocellulose blotting membranes. We use 0.45 μm pore size for immunoblotting Rb, but a smaller pore size may be recommended should you want to adapt this protocol for low molecular weight proteins. Choose membrane pore size according to the molecular weight of the protein of interest.

2. 1X transfer buffer: Dissolve 3.03 g of Tris base and 14.4 g of glycine in 500 ml of H₂O. Add 200 ml of methanol, and complete to a final volume of 1 L with H₂O.

3. Tris-buffered saline with Tween-20 (TBST): First prepare a 10X TBS stock by dissolving 24.2 g of Tris base and 87.6 g of NaCl in 800 ml of H₂O, adjust to pH 7.6 with 1 M HCL, and complete to a final volume of 1 L. To prepare the TBST, add 1 ml of Tween-20 to 1 L of 1X TBS.

4. Ponceau-S membrane staining solution. We recommend that you use this dye to stain the membrane after transfer to verify the presence of protein in the membrane. This gives an indication of how effective the transfer was. Prepare a 0.5% (w/v) of Ponceau-S in a 1% acetic acid solution. To remove the Ponceau-S stain from the membrane before blotting, you need TBST with 5% dry milk.

5. Filter paper.

2.5. Immunoblotting Reagents

The protocols described in this chapter were optimized specifically for the antibodies described below and using lung cancer cell lines. The protocol can be adaptable to other phospho-specific antibodies, but additional optimization could be required, specifically in the antibody dilution and incubation times.

1. Blocking solution. Dissolve 0.5 g of bovine serum albumin (BSA) in 10 ml of 1X TBST.

2. Primary antibody against phosphorylated serine 249 in Rb (anti-Rb Phospho-Ser249). We purchase this rabbit polyclonal antibody from Sigma Aldrich (Cat. No. SAB1305397) and use it at a dilution of 1:500 in TBST. We usually prepare primary antibody solutions in TBST that can be stored for several months at 4°C. To prepare such antibody solutions, first dissolve 0.5 g of BSA in 10 ml of TBST and add 30 μl of a 20% Sodium azide stock solution. Mix well and add the antibody at the indicated dilution (see Note 4).

3. Primary antibody against total Rb (mouse monoclonal 4H1, Cell Signaling Cat. No. 9309). In order to validate a phosphor-antibody, you need to blot the protein lysate with both antibodies, one against the phosphorylated form of the protein and the other against the total protein. We use this antibody at a dilution of 1:1000 in TBST, and we prepare it exactly as described above for the antibody against phospho-S249. It is important to blot for total Rb, as the extent of Rb phosphorylation is assessed as the ratio of phosphorylated Rb to total Rb protein.

4. Secondary antibodies: we use horse-radish peroxidase (HRP)-conjugated secondary antibodies. For mouse monoclonal primary antibodies, we use an HRP-conjugated, affinity-purified horse anti-mouse IgG (Cell Signaling, Cat. No. 7076S). For rabbit polyclonal primary antibodies, we use an HRP-conjugated, affinity-purified goat anti-rabbit IgG (Cell Signaling, Cat. No. 7074S). For both of these secondary antibodies, we prepare a TBST solution of the antibody exactly as described above for primary antibodies (except that we omit the sodium azide since we prepare fresh for each use), with the antibody diluted to 1:5,000.

5. Supersignal West Pico Plus^™ Chemilumiscent Kit. This kit is compatible with HRP-conjugated secondary antibodies. The selection of the kit to develop the chemiluminescent signal is dictated by the enzyme conjugated to the secondary antibody (HRP, versus alkaline phosphatase, for example). Other available kits are acceptable, provided they are compatible with your choice of antibodies. Use strictly following the kit’s instructions.

6. ChemiDoc imaging system and software, or other equivalent imaging system compatible with chemiluminescent signals.

2.6. Additional Laboratory Equipment and Plasticware

1. Tabletop centrifuge with capacity for 15 ml tubes, preferably refrigerated.

2. Conical tubes, 15 and 50 ml volumes.

3. Culture plates or bottles, 35 mm or other of your preference.

4. Gel electrophoresis system, including power source. Assemble and use as per manufactureŕs instructions.

5. Transfer system, including power source. Assemble and use as per manufactureŕs instructions.

6. 1.5 ml microcentrifuge tubes.

7. Refrigerated centrifuge for microcentrifuge tubes.

8. Ice bucket, ice.

9. Heat plate, with capacity to hold 1.5 ml microcentrifuge tubes. To be used at 30°C, 70°C, and 95–100°C.

10. Glass or plastic Pasteur pipettes.

3. Methods

3.1. Cell Lysis and Preparation of Protein Extracts

1. Ensure that you start with cells cultured at approximately 90–95% confluence. Collect cells by scraping them from the culture plate in 1–2 ml of 1X PBS. We culture cells in 35 mm culture plates, you should adjust the volume of PBS depending on your culture plate, but it is important to use the minimum volume of PBS to cover the entire plate surface with a thin PBS layer. Alternatively, detach cells from the plate by incubating in trypsin-EDTA solution at 37°C for 5 minutes (see Note 5).

2. Transfer the cell suspension to a 15 ml tube and pellet cells by low speed centrifugation (5 min at 300–400 × g). If you detach the cells using the trypsin-EDTA solution, before the centrifugation step you need to dilute it 1:10 with culture medium to ensure inactivation of trypsin. Remove the supernatant after the centrifugation.

3. Lyse cells by resuspending the cell pellet in RIPA buffer supplemented with the protease inhibitor cocktail (ensure that the cocktail does not include phosphatase inhibitors, since these will inhibit subsequent steps). Use the minimal possible volume of RIPA buffer to ensure adequate protein concentration in the lysate. Transfer the cell suspension to a 1.5 ml microcentrifuge tube.

4. Incubate at 4°C or on ice for 30 min to allow lysis to proceed.

5. Centrifuge tube for 10 min at 1,400 ×g at 4 °C. Transfer supernatant to a fresh microcentrifuge tube.

6. Quantify the protein in your cell lysate using your method of choice, using a BSA concentration curve to determine protein concentration in your sample (see Note 6).

3.2. Protein Lysate Dephosphorylation with Alkaline Bovine Intestinal Phosphatase (BIP)

1. Once the amount of protein in the lysate has been quantified, proceed to prepare the de-phosphorylation reaction in a 1.5 mL microcentrifuge tube, following the indications shown in Table 3. Notice that you need to prepare three reactions for each protein lysate you wish to analyze: one reaction with protein lysate with only the BIP buffer (this acts as a negative control since the phosphorylation of lysate proteins should remain unaffected); a second reaction with protein lysate, BIP buffer and the BIP enzyme; and a third reaction to which you will add the BIP inhibitor cocktail in addition to the components of the second reaction. You can de-phosphorylate between 400–500 μg of total protein with 100 Units of BIP [10–13]. Given the 123 U/ μL activity of the BIP enzyme we use, we can use 1 μL of enzyme to treat 400–500 μg of total protein. You can prepare a final reaction volume of 50 μL. It is recommended that you aim to obtain highly concentrated protein extracts (at least 50 μg/μL) in order to be able to perform the reaction in such a small volume.

2. Incubate reaction mixtures for 30 min at 30°C (if using the Sigma-Aldrich BIP), or as indicated by the manufacturer if using a BIP from other vendors (see Note 7).

3. Stop the reaction by transferring the reaction mixture to ice.

4. Take an aliquot from each reaction mixture, the volume should contain 20–30 μg of total de-phosphorylated protein. Mix with an equal volume of 2X SDS-PAGE sample loading buffer. The remaining reaction mixture can be stored at −20°C.

5. Denature proteins by heating the sample at 95–100 °C for 5 min, or at 70 °C for 30 min. Proceed to the SDS-PAGE separation of the sample described in the next section.

Table 3.

Amounts of the components used in the set-up of the BIP de-phosphorylation reaction

Reaction	Amount of Protein (400–500 μg)	Amount of 10X dephosphorylation buffer	Amount of Phosphatase (123 Units/uL)	Amount of 2X Phosphatase Inhibitor	H2O
Protein Lysate + Buffer	10 μL	5 μL	---------	----------	35 μL
Protein Lysate + Buffer + BIP*	10 μL	5 μL	1 μL	----------	34 μL
Protein Lysate + Buffer + BIP* + BIP Inh.**	10 μL	5 μL	1 μL	25 μL	9 μL

^*BIP = Bovine Intestinal Alkaline Phosphatase;

^**BIP Inh. = Bovine Intestinal Alkaline Phosphatase Inhibitor. Final reaction volume can be 50 μL. Try to obtain concentrated protein lysates with a protein concentration of at least 50 μg/ μL. This will allow you to use a volume of approximately 10 μL in the final 50 μL reaction

3.3. SDS Polyacrylamide Gel Electrophoresis and Transfer

1. Assemble your gel electrophoresis apparatus following manufactureŕs instructions. At this point you will only need to assemble the gel casting system needed to pour the gels. We use a standard Bio rad gel electrophoresis apparatus with its accompanying gel casting system. Be sure to clean thoroughly all glass plates with 70% ethanol. This will decrease the risk of forming air bubbles while pouring the gel into the glass plates.

2. Prepare the separating gel as follows: in a 50 ml conical tube, mix 7.9 ml of distilled H₂O, 7 ml of the 30% acrylamide, bis-acrylamide mix, 5.0 ml of 1.5 M Tris HCl pH 8.8 and 0.2 mL of 10% SDS. Add then 200 μl of 10% APS and 8 μl of TEMED. Gently mix avoiding the formation of bubbles. The gel will start rapidly polymerizing after the addition of APS and TEMED, therefore these two reagents should be the very last to be added to the mix, and the gel should be poured immediately after their addition. Maintaining the mix on ice will retard the polymerization (see Notes 8 and 9).

3. Allow the gel to completely polymerize for 30–60 min at room temperature (see Note 10). Be sure to always use freshly prepared or thawed APS (avoid re-freezing of leftovers, discard them). Loss of APS activity is manifested in abnormally long polymerization times.

4. Prepare the upper stacking gel as follows: in a 15 ml conical tube, mix 4.1 ml of distilled H₂O, 1.0 mL of 30% acrylamide/bis-acrylamide mix, 750 μl of 0.5 M Tris-HCl pH 6.8, and 60 μl of 10% SDS. Mix gently avoiding foam and bubbles. When ready to pour, add 60 μl of 10% APS and 6 μl of TEMED. Mix and add it to the glass plates (see Note 11).

5. Immediately after pouring the stacking gel, insert comb being careful not to form any bubbles at the base of the wells. Allow the stacking gel to polymerize for 30–60 min at room temperature.

6. Carefully remove the comb and the bottom spacer. We do not recommend that you remove the comb straight out of the dry gel. Rather, we recommend that first you assemble the whole electrophoresis apparatus, including inserting the gel inside it, fill the liquid reservoir with running buffer, and then remove the comb. We use a standard protein gel electrophoresis apparatus from Bio-Rad. Dilute the 10X SDS PAGE running buffer to 1X with distilled water and fill the assembled apparatus with it until you cover the gel. Only then we recommend that you slowly and carefully remove the comb (see Note 12). Using a glass or plastic Pasteur pipette, rinse the wells with running buffer to remove excess acrylamide.

7. After denaturing the protein samples as indicated in Section 3.2, steps 4 and 5, load them into the wells, being careful not to over flood the wells (if this happens, you risk having a protein sample over flooding into an adjacent lane). Remember also to load the protein ladder.

8. Place the lid on the electrophoresis apparatus, connect to a power supply, and run the gel at 160 V for 60 min (do not set a limit for Amperes). Monitor the run by following the bromophenol blue dye (from the sample loading buffer) front. Stop the run when the dye front reaches about two-thirds of the length of the frontal glass plate.

3.4. Transfer of Proteins to Nitrocellulose Membranes

1. Disassemble the electrophoresis apparatus and remove the gel assembly. Very gently and carefully separate the glass plates from the gel inserting a fine spatula in between the glass plates, and slowly twisting the spatula until the plates start to separate from the gel. Use a razor blade to carefully remove the stacking gel without damaging the separating gel. Rinse the gel with transfer buffer, keep it submerged in transfer buffer, never let the gel dry.

2. Cut a piece of nitrocellulose membrane of the approximate size of the gel (a little bit larger so you can handle it by the edges without touching the gel) and immerse in cold transfer buffer for 2–5 min. Always handle the membrane with gloves or with tweezers. Never touch the membrane with bare hands, this may leave fingerprints oils on the membrane and this in turn will prevent even wetting of the membrane. This usually results in areas in the membrane where transfer of proteins is impaired.

3. Pre-wet several pieces of filter paper (cut in a size similar to the gel) in cold transfer buffer by submerging one side of the paper first and then slowly lowering it into the buffer.

4. Assemble the gel electro-transfer cassette following the manufacturers’ instructions. Avoid bubbles being trapped between the gel and the membrane, as protein transfer will not occur adequately at these sites.

5. Insert the transfer cassette into the electro-transfer unit. It is usual for electro-transfer units to have a special compartment for an ice block or any other cooling device. As the transfer process generates heat and the transfer buffer can get warm (or even hot), it is recommended that the ice block/cooling devices are used. The transfer can be done in a cold room, or the whole transfer apparatus can be inserted in a tray and surrounded with ice during the transfer process.

6. Transfer for 60 min at 100V (do not set a limit for Amperes).

7. After the transfer is completed, disassemble the unit. Wash the membrane in TBST to remove residual SDS and potential gel fragments.

8. Check the efficiency of transfer by staining the membrane in Ponceau-S solution for 5 min at room temperature. Record an image of the Ponceau-stained membrane using a document scanner or camera. Even transfer (no air bubbles) of equal amount of proteins per lane should be seen in the Ponceau-stained membrane. Never allow the membrane to get dry. Keep it moist during the documentation process by wrapping it in plastic wrap after soaking in transfer buffer. See Notes 13, 14 and 15 for transfer troubleshooting tips.

3.5. Immunoblotting, Image Development and Capture

1. Remove the Ponceau-S staining from the membrane by incubating in TBST containing 5% milk. You will notice the milk solution turning red. Discard and rinse the membrane with TBST (no milk). Repeat until no trace of the red Ponceau-S stain remains in the membrane. At this point you should only see the rainbow-colored protein markers. Repeat a final rinse with TBST (see Note 16).

2. Block membranes in TBST with BSA (see Note 16). You can block for 2 h at room temperature or overnight at 4°C. Use constant rotation to ensure that the membrane is constantly bathed by the solution.

3. Incubate membranes with the primary antibody. Like the blocking step, primary antibody incubation can be done either 2 h at room temperature, or overnight at 4°C. Use constant motion during this step as well (see Note 17).

4. Wash three times in TBST.

5. Incubate with secondary antibody for 1 h at room temperature while agitating.

6. For development of the membrane chemiluminescence, we use the Supersignal West Pico Plus^™ Chemilumiscent Kit (Thermo Scientific Cat. No. 34580), following the procedures exactly as described by the manufacturer. We add 1 ml of substrate solution per membrane. Do not leave it for more than 3 minutes (see Note 18).

7. Capture the chemiluminescent signal using a ChemiDoc imaging system or its equivalent (we use ChemiDoc XRS+), using the accompanying image software for image capturing and quantification of signal intensity.

3.6. Interpretation of Results

We usually run in parallel immunoblots using the phosphor-specific antibodies as well as antibodies recognizing total Rb protein. You need to assess phosphorylation of your protein of interest in particular residues, relative to the total amount of that specific protein. In our case, we document protein Rb phosphorylation as the ratio of phosphorylated Rb to total Rb [9, 14]. When using an antibody against the total protein, usually phospho-proteins can be appreciated in western blots as a doublet consisting of two bands migrating close to each other [9, 14]. In that doublet, the upper band usually corresponds to the phosphorylated form of the protein (as phosphorylation increases the protein’s molecular weight), while the lower band corresponds to the unphosphorylated form. Alternatively, if a doublet is not apparent, the phosphorylated form may appear as a single band but of higher molecular weight relative to the unphosphorylated form. You should still be able to see this doublet (or the higher weight band) in the sample treated with BIP buffer alone, since this is your negative control reaction. However, in the sample treated with BIP, the doublet should disappear (only the lower band should remain), or the higher molecular weight form should be displaced to a lower molecular weight position in the gel. When using the antibody that recognizes only the phosphorylated form, BIP treatment should eliminate immunoreactivity of the slower migrating higher molecular weight bands, confirming that these bands correspond to phosphorylated versions of the protein. This change is indicative of the removal of the phosphate groups from the protein, which translates into a faster electrophoretic mobility. This change should be reversed when the sample is treated with both the BIP and the phosphatase inhibitor cocktail, so that the band pattern in this sample is comparable to untreated samples or to samples treated with BIP buffer alone without enzyme.

4. Notes

1. Protease inhibitors in the cell lysis buffer, combined with maintaining the protein lysate on ice at all times, minimize protein degradation during extraction and handling of the lysate. We use a broad specificity protease inhibitor cocktail from Sigma-Aldrich (Cat. No. P8340–5ML). Other alternatives are acceptable, follow manufacturer’s instructions regarding working concentration and handling. Be sure that the inhibitor cocktail does not contain phosphatase inhibitors, as these will inhibit BIP activity.

2. We use the pre-mixed Bio-Rad acrylamide solution. Store at 4 °C. Please be aware that polyacrylamide is toxic. Carefully read the accompanying Materials Safety Data Sheet for specific instructions on how to handle and dispose polyacrylamide solutions.

3. Make small volume aliquots and store at −20 °C. Avoid repeated freezing and thawing cycles. Do not use leftovers for future experiments, a fresh aliquot should be thawed for each experiment.

4. Sodium azide is used as a preservative to prevent bacterial growth in the solution. This is highly recommended if you plan to reuse the primary antibody solution and store it for long term use. This solution can be stored and reused for several months, but you need to be attentive for signs of bacterial and fungi contamination such as a strong odor or cloudiness in the solution. In such case, discard and prepare a fresh solution. Use of contaminated antibody solution usually yields high background in western blots, meaning that it is time to replace the solution with a fresh one.

5. Avoid over-trypsinization as it may kill cells. Fresh trypsin solution should detach cells in under 5 minutes. If you find that 5 minutes under trypsin-EDTA are not enough to detach cells from the plate, it is better to get a fresh trypsin-EDTA batch than prolonging the trypsinization time. Avoid trypsin when studying membrane proteins.

6. We perform a standard protein quantification assay using Bradford Assay, following its instructions, and using BSA as a quantification standard. Other substitute assays can be used. After quantification, protein lysates that will not be used immediately can be stored at −20°C for up to 3 months (protein integrity cannot be ensured beyond that).

7. This treatment should eliminate immunoreactivity if the antibody is indeed recognizing a phosphorylated form of the protein of interest. This will be observed after western blot evaluation. Remember also run in the SDS-PAGE the controls treated with phosphatase buffer alone, as well as a control with phosphatase in the presence of phosphatase inhibitors.

8. It is recommended that you add a thin layer of isopropanol on top of the separating gel solution immediately after pouring it. Isopropanol is not miscible in water thus it will form a distinct layer. Adding isopropanol eliminates any bubbles on the surface of the separating gel solution and will produce a smooth surface.

9. For Rb, which has a molecular weight of approximately 110 kDa, we use a 10% polyacrylamide separating gel. The % of polyacrylamide you will choose depends on the molecular weight range in which you want to have good resolution. Take this into consideration if you wish to adapt this protocol to other phosphor-proteins.

10. After 45 minutes, you can verify if the gel has polymerized by gently tilting the casting apparatus sideways. Only the isopropanol layer should move while the underlying separating gel should be static if it has polymerized.

11. To save time, you can start preparing the stacking gel while the separating gel is polymerizing. However, do not add the TEMED and the APS until immediately before adding the stacking gel to the casting apparatus. The stacking gel without APS and TEMED can be kept on ice until pouring. The % of acrylamide of the stacking gel is usually smaller (4–6%) than that of the separating gel.

12. If you slowly remove the comb having the gel submerged in running buffer, you will notice that as you remove the comb the empty well space is immediately filled with buffer. This will avoid the collapse of the well that is experienced if you remove the comb out of the dry gel, as a vacuum is formed inside the well as the comb is retrieved.

13. Do not exceed the transfer time as this leads to gel shrinkage and distortion of the membrane. In case of poor transfer efficiency, opt for making thinner gels, rather that prolonging transfer time.

14. If there are unstained “white spots” on the membrane seen after Ponceau-S staining, this may have been caused by air bubbles trapped between the gel and the membrane. Make sure to remove all the bubbles when preparing the transfer cassette. An air bubble does not necessarily ruin an experiment, it depends on its size and on the molecular weight range in which it formed. If your protein of interest is not in this range, you may choose to proceed with the subsequent steps.

15. Ponceau-S staining may also help you to spot degraded proteins, which are appreciated as a diffuse smear in the lower half on the membrane. In this case, ensure that you are taking all the precautions necessary to deal with protein degradation, such as not using protein samples that have been stored for prolonged times (or repeatedly frozen-thawed), ensuring that cell lysis was done on ice and the samples were kept cold or refrigerated all the times, and that you added protease inhibitors to the RIPA buffer.

16. When doing a western blot using antibodies against phosphorylated residues, it is important that you thoroughly remove any traces on milk from the membrane. Casein (milk protein) is heavily phosphorylated and any traces of milk in the membrane can lead to high background due to non-specific antibody binding. For the same reason, the blocking solution must not contain milk. The blocking step is usually one of the steps in which you can make adjustments in case you experience high background levels.

17. Longer incubation periods are recommended if you are having trouble obtaining strong signals. However, be aware that longer incubation times also increase the likelihood of obtaining a strong background. The length of the incubation time (with primary antibody) is one of the factors that affect signal strength. Dilution of antibody also usually affects background noise. If you are obtaining too much background, in addition to extending blocking time, you can try diluting the primary antibody. Conversely, try concentrating the primary antibody if you obtain weak signals.

18. This step can be performed for 30 seconds to 3 minutes. If you are experiencing weak signals, in addition to using more concentrated primary antibody and/or increasing incubation time, you can try developing the membrane for longer (but do not exceed 3 minutes, as this may blacken the membrane). Some antibodies give a very strong signal and in such cases 30 seconds to 1 minute is sufficient.