Western Blot Protocol & Troubleshooting Guide
The ultimate Western blot guide
Everything you need for a successful Western blot in one place — a complete 14-step protocol with every buffer recipe, plus 101 troubleshooting fixes for weak signal, high background, non-specific bands, poor transfer and more.
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What is Western blotting?
Western blotting determines the presence or absence of selected proteins in a sample. It is advantageous over other antibody-based assays such as ELISA because cross-reactivity with non-target proteins can be distinguished from the target based on molecular weight.
Proteins are first separated by size using gel electrophoresis, then transferred to a nitrocellulose or PVDF membrane by an electrical current. The membrane is probed with antibodies specific for the protein of interest, giving qualitative or semi-quantitative information.
Western blot principle
| Step | Procedure |
|---|---|
| 1 | Extract proteins from cells or tissue with a detergent lysis buffer, then centrifuge to remove debris. |
| 2 | Separate proteins by size using gel electrophoresis (agarose or polyacrylamide). |
| 3 | Transfer proteins to a nitrocellulose or PVDF membrane using transfer buffer and an electric current. |
| 4 | Detect the target with a specific primary antibody, then a labelled secondary antibody (fluorophore or enzyme). |
| 5 | For PVDF, re-activate the membrane with methanol, then wash again in TBST. |
The 14-step Western blot protocol
A complete, laboratory-tested protocol from lysate to imaged membrane, with every buffer recipe you need along the way.
1Protein extraction
The protein of interest must be solubilised to migrate through the gel. Choice of lysis buffer depends on the protein yield required and the subcellular localisation of the target. SDS and other ionic detergents give the highest yield but are the most denaturing; milder non-ionic detergents (NP-40, Triton X-100) are used when the antibody only recognises native protein. Where protein-protein interactions must be preserved, use a buffer without ionic or non-ionic detergents (e.g. mechanical shearing).
| Protein localisation | Recommended buffer |
|---|---|
| Cytoplasmic (cytoskeletal-bound) | Tris-Triton |
| Cytoplasmic (soluble) | Tris-HCl |
| Membrane-bound | NP-40 / RIPA |
| Mitochondria | RIPA |
| Nuclear | RIPA |
| Whole cell | NP-40 / RIPA |
1.1 Extraction buffer recipes
RIPA buffer
- 50 mM Tris-HCl pH 7.4
- 50 mM NaCl
- 2 mM EDTA
- 1% SDS
- + fresh protease inhibitors (aprotinin, leupeptin, DTT, PMSF)
NP-40 buffer
- 150 mM NaCl
- 1.0% NP-40 (or Triton X-100)
- 50 mM Tris pH 8.0
Tris-HCl buffer
- 20 mM Tris-HCl pH 7
Tris-Triton buffer
- 10 mM Tris pH 7.4
- 100 mM NaCl
- 1 mM EDTA
- 1 mM EGTA
- 1% Triton X-100
- 10% glycerol
- 1% SDS
- 5% deoxycholate
Laemmli buffer
- 4% SDS
- 10% 2-mercaptoethanol
- 20% glycerol
- 0.004% bromophenol blue
- 0.125 M Tris-HCl, pH 6.8
All buffers can be stored at 4°C for several weeks and at −20°C for up to a year.
1.2 Protease & phosphatase inhibitors
Once cell lysis begins, protein degradation begins too. Keep samples on ice at all times and add fresh inhibitors to the lysis buffer each time.
| Inhibitor | Target |
|---|---|
| Aprotinin | Chymotrypsin, plasmin, trypsin |
| EDTA | Metalloproteases (Mg²⁺/Mn²⁺) |
| EGTA | Metalloproteases (Ca²⁺) |
| Leupeptin | Lysosomal proteases |
| NaF | Ser/Thr phosphatases |
| Na orthovanadate | Tyrosine phosphatases |
| Pepstatin A | Aspartic proteases |
| PMSF | Serine, cysteine proteases |
1.3 Cell-lysate preparation with RIPA
| Step | Procedure |
|---|---|
| 1 | Wash cells with ice-cold PBS. |
| 2 | Aspirate the PBS. |
| 3 | Add ice-cold RIPA buffer (~1 mL per 10⁷ cells). |
| 4 | Scrape adherent cells off the plate with a sterile tip. |
| 5 | Centrifuge (force/time depend on cell type). |
| 6 | Remove from the centrifuge and store on ice. |
| 7 | Aspirate supernatant into a fresh tube on ice; discard the pellet. |
| 8 | Determine protein concentration by Bradford, Lowry or BCA (BSA standard). |
| 9 | Freeze at −20/−80°C or prepare for loading. |
1.4 Preparing samples for loading
If the antibody recognises native protein, do not denature the sample and leave SDS and reducing agents (β-mercaptoethanol, DTT) out of the loading and migration buffers. For antibodies requiring denatured protein, heat the sample in a denaturing loading buffer (e.g. SDS) at 95–100°C for 5 minutes. The standard loading buffer is 2× Laemmli buffer, which contains a reducing agent, SDS for negative charge, glycerol for density and bromophenol blue as a migration front.
2Electrophoresis
With the sample lysed, quantified and loading buffer added, proteins are separated by SDS-PAGE. A gel has a resolving portion (poured first) and a stacking portion (poured on top, holding the combs). Higher acrylamide percentages give smaller pores for small proteins; lower percentages suit larger proteins.
| Acrylamide % | Linear separation range (kDa) |
|---|---|
| 5.0 | 57–211 |
| 7.5 | 36–94 |
| 10 | 16–68 |
| 15 | 12–43 |
2.1 Gel recipes
10% resolving gel
- H₂O 5.9 mL
- 30% acrylamide-bis 5 mL
- 1.5 M Tris pH 8.8 3.8 mL
- 10% SDS 150 µl
- 10% APS 150 µl
- TEMED 6 µl
Stacking gel
- H₂O 2.7 mL
- acrylamide-bis 670 µl
- Tris pH 6.8 500 µl
- 10% SDS 40 µl
- 10% APS 40 µl
- TEMED 3 µl
3Controls & molecular-weight markers
Use positive and negative controls to confirm the assay is accurate, sensitive and efficient, and a loading control to confirm even loading. A range of molecular-weight markers lets you determine protein size and monitor the run.
| Sample type | Protein | MW (kDa) |
|---|---|---|
| Whole cell / cytoplasmic | β-actin | 43 |
| α-actin | 43 | |
| GAPDH | 30–40 | |
| β-tubulin | 55 | |
| α-tubulin | 55 | |
| High MW | vinculin | 116 |
| Mitochondria | VDAC1/porin | 31 |
| cytochrome C oxidase | 16 | |
| Nuclear | lamin B1 | 66 |
| TATA-binding protein (TBP) | 38 | |
| PCNA | 29 | |
| Serum | transferrin | 77 |
| Muscle | SDHA | 73 |
4Loading samples & running the gel
Separate proteins by SDS-PAGE at an acrylamide percentage suited to your target. A worked example using a 10% gel:
| Step | Procedure |
|---|---|
| 1 | Pour the resolving gel between the plates; let it set for 1 h. |
| 2 | Level with ~300 µl isopropanol poured gently across the top. |
| 3 | Remove isopropanol with a gentle flow of water. |
| 4 | Add the stacking gel and insert the comb, avoiding bubbles; set for 1 h. |
| 5 | Place the plate in the rig and add ~400 mL of 1× SDS-PAGE running buffer, checking for leaks. |
| 6 | Remove the combs and rinse the wells with running buffer. |
| 7 | Load 8 µl of unstained MW ladder and 20 µl of each protein sample. |
| 8 | Run at 25 mA per gel for ~1 h 15 min. |
5Western blot transfer
After electrophoresis, transfer the protein from gel to membrane (nitrocellulose or PVDF), most commonly by electrophoretic transfer. The gel is placed in direct contact with the membrane between two electrodes in a conducting solution; applying a field migrates proteins onto the membrane, creating a copy of the gel pattern.
Tip: always probe for your weakest antibody first, as handling and stripping can unstick your proteins of interest.
5.1 Transfer buffer
Transfer buffer
- 8 g Trizma base
- 9 g glycine
- 3.7 g SDS
- 200 mL methanol
- dH₂O to 1 L
6Blocking
Block the membrane before adding antibody to prevent non-specific binding and improve the signal-to-noise ratio. Milk and BSA are most common; use BSA with phosphorylation-sensitive antibodies. If the primary was raised in horse, cow, goat or donkey, avoid BSA/milk due to cross-reaction or IgG contamination.
Blocking buffer
- 5% marvel (milk) in TBS-Tween
- (1× TBS with 0.1% v/v Tween-20)
7Wash buffers
Wash steps remove unbound reagents and reduce background. Too little washing raises background; too much can elute the antigen. Tween-20 is typically 0.05–0.5%. Make fresh detergent stocks (microbial growth raises background) and use high-purity detergent (peroxides interfere).
Wash buffer (TBS-T)
- 20 mM Tris pH 7.5
- 150 mM NaCl
- 0.1% Tween-20
8Primary antibody incubation
Probe the blocked membrane with a primary antibody specific for your target. Consider species specificity, sensitivity to denaturing conditions and post-translational modifications, and whether the antibody is validated for Western blot. After incubation, wash 5× for 5 minutes with wash buffer.
9Secondary antibody incubation
Because the primary is not directly detectable, a labelled secondary that binds the primary is used. Match the secondary to the host species of the primary (or its tag, e.g. biotin, histidine). Secondaries are conjugated to HRP or AP for film/chemiluminescence, or to a fluorophore for laser detection.
10Antibody dilution
Manufacturers recommend a starting dilution, but the optimum varies with the assay. Strongly expressed targets and highly sensitive assays need less antibody; weakly expressed targets need more. Less antibody decreases background and increases specificity. Dilutions are normally made in wash buffer.
11Blot development
The secondary label determines the development system: chemiluminescence for HRP/AP, or direct fluorescent scanning for fluorophores.
11.1 Chemiluminescence
HRP conjugates are most widely used and generally superior to AP (smaller enzyme, high activity, stable, widely available substrates). The signal is transient; a well-optimised assay produces light for 1–24 h for film or digital imaging. Digital imaging gives a broad dynamic range and quantitative data; chemiluminescent signal is generally semi-quantitative.
11.2 Fluorescent detection
Fluorophore conjugates need fewer steps (no substrate) and a shorter protocol, but require an excitation source. Infrared/near-infrared and quantum dots have improved sensitivity. Fluorescence enables multiplexing and quantitative, consistent results, though auto-fluorescence can reduce signal-to-noise.
11.3 Chemiluminescent vs fluorescent
| Chemiluminescent | Fluorescent | |
|---|---|---|
| Principle | Enzyme label (HRP/AP) | Fluorophore label |
| Detection | X-ray film / digital imaging | Laser scanning imager |
| Multiplex | No | Yes |
| Signal duration | Hours | Weeks to months |
| Linear dynamic range | 15-fold (film); 3–4,000-fold (digital) | >4,000-fold |
| Quantitation | Semi-quantitative | Quantitative |
| Substrate | Luminol | None needed |
12Stripping & re-probing
Stripping removes primary and secondary antibodies so you can probe for another target (e.g. a loading control), saving time and sample. It works best on PVDF. Chemiluminescent reagents like ECL are recommended as they don’t stain the membrane. After stripping, wash thoroughly and re-block before the next primary.
12.1 Mild vs harsh
A mild buffer uses low-pH glycine to dissociate antibodies. A harsh buffer (for high-signal blots) uses low pH plus reducing agents (β-mercaptoethanol, SDS) heated at 50–80°C for up to 45 min; its effect is irreversible, so wash thoroughly before re-probing.
Mild stripping buffer
- 15 g glycine
- 1 g SDS
- 10 mL Tween-20
- dissolve in 800 mL dH₂O
- adjust to pH 2.2
- bring to 1 L
Harsh stripping buffer
- 62.5 mM Tris-HCl pH 7.8
- 100 mM β-mercaptoethanol
- 2% (w/v) SDS
12.2 Stripping protocol
| Step | Procedure |
|---|---|
| 1 | Incubate PVDF with harsh stripping buffer 30 min at 50°C. |
| 2 | Wash 2×5 min, 1×10 min, 2×5 min at RT with agitation. |
| 3 | Block 1 h at RT with gentle agitation. |
| 4 | Wash 2×5 min, then incubate with the appropriate primary and secondary. |
13Protein gel visualisation
Gel separation can be visualised by Coomassie or copper staining, depending on downstream use.
13.1 Coomassie staining
Confirms uniform, even migration. Use only if you do not intend to transfer, as Coomassie staining is not reversible.
| Step | Procedure |
|---|---|
| 1 | Treat the gel with 40% dH₂O, 10% acetic acid, 50% methanol to precipitate proteins. |
| 2 | Add 0.25% (w/w) Coomassie blue to the same solution. |
| 3 | Incubate 4–24 h at RT on a shaker. |
| 4 | Rinse in 67.5% dH₂O, 7.5% acetic acid, 25% methanol. |
| 5 | Replace with fresh rinse once excess dye is removed. |
| 6 | Excess stain washes out, leaving a clear gel. |
| 7 | Stain binds proteins, giving blue bands. |
13.2 Copper staining
Use if you wish to transfer afterwards — faster and more sensitive than Coomassie.
| Step | Procedure |
|---|---|
| 1 | Briefly rinse the gel in ddH₂O after electrophoresis. |
| 2 | Transfer to 3 M CuCl₂ for 5–15 min. |
| 3 | Wash in ddH₂O. |
| 4 | View against a dark-field background. |
| 5 | Protein appears as clear zones on a translucent blue background. |
| 6 | De-stain in 0.1–0.25 M Tris / 0.25 M EDTA pH 8.0. |
| 7 | Place in transfer buffer and proceed. |
14Protein membrane visualisation
Assess transfer efficiency by staining the PVDF/nitrocellulose membrane with Ponceau Red. It is easily reversed by washing, so it does not interfere with subsequent antibody probing.
| Step | Procedure |
|---|---|
| 1 | Dilute Ponceau stock 1:100. |
| 2 | Incubate on an agitator for 5 min. |
| 3 | Wash with ddH₂O until the water is clear and bands are visible. |
| 4 | De-stain by washing with TBST. |
| 5 | For PVDF, re-activate with methanol then wash again in TBST. |
101 Western blot troubleshooting tips
No bands, faint signal, non-specific or wrong-size bands, poor transfer, high background — find the likely cause and fix fast. Filter by symptom or search your problem.
| Symptom | Likely cause | Fix |
|---|---|---|
| No bands | Incorrect primary antibody | Antibody has low to no affinity for the target. |
| No bands | Inactive antibody | Perform a dot blot to confirm antibody activity. |
| No bands | Insufficient protein concentration | Increase the amount of protein loaded and use a positive control. |
| No bands | Poor transfer | Ensure the membrane is activated. Transfer buffer must contain methanol for nitrocellulose; PVDF must be pre-soaked in methanol. |
| No bands | Incomplete transfer | Stain the membrane with India Ink, Amido Black or Ponceau S to check transfer. |
| No bands | Over-transfer | Reduce transfer time or voltage. |
| No bands | Incorrect secondary antibody | Confirm the host species and IgG type of the primary. |
| No bands | Antibodies expired | Check that all antibodies are in date. |
| No bands | Incorrect antibody storage | Store all antibodies per manufacturer instructions. |
| No bands | Suboptimal primary incubation time | Increase incubation time with the primary antibody. |
| No bands | Incompatible primary and secondary | Maintain a consistent species across both antibodies. |
| No bands | Insufficient secondary concentration | Increase the concentration of primary/secondary antibody. |
| No bands | Excessive washing | Reduce the number and duration of washes. |
| No bands | Incorrect orientation | Mark your membrane to ensure correct orientation. |
| No bands | Contaminated wash or incubation buffer | Use fresh, sterile buffer. |
| No bands | Insufficient exposure time | Re-image the blot with a longer exposure time. |
| No bands | Incorrect filter settings | Ensure the detection instrument reads the correct wavelengths. |
| No bands | Reduced antibody efficacy from overuse | Use fresh primary and secondary antibodies each experiment. |
| No bands | Absence of protein of interest | Run a positive control. |
| No bands | Sodium azide contamination | Sodium azide quenches HRP signal — remove it. |
| No bands | Isoelectric point >9 | Use a higher-pH buffer system, e.g. CAPS (pH 10.5). |
| Faint bands | Insufficient antibody concentration | Increase antibody concentration 2–4 fold over starting. |
| Faint bands | Insufficient protein concentration | Increase total protein loaded on the gel. |
| Faint bands | Suboptimal antibody binding | Reduce washes; reduce NaCl in blotting/antibody solution (0.15–0.5 M). |
| Faint bands | Inactive conjugate | Purchase new reagents or switch to ECL. |
| Faint bands | Old or weak ECL | Use new ECL reagents. |
| Non-specific | Non-specific antibody | Ensure the antibody is specific for the protein of interest. |
| Non-specific | Proteolytic breakdown | Use protease inhibitors to prevent breakdown of the antigen. |
| Non-specific | Gel overloading | Too much protein causes ghost bands — optimise protein amount. |
| Non-specific | Insufficient blocking | Extend the blocking time. |
| Non-specific | Low antigen concentration | Consider immunoprecipitating the target protein. |
| Non-specific | Non-specific secondary binding | Run a secondary-only control; if bands appear, switch secondary. |
| Non-specific | Analyte aggregation | Increase DTT concentration. |
| Non-specific | Analyte degradation | Make fresh samples; reduce freeze/thaw; add protease inhibitors before storage. |
| Non-specific | Protein degradation | Target protein has degraded — prepare fresh sample. |
| Non-specific | Splice variants | May lead to multiple bands. |
| Non-specific | High primary concentration | Use a lower concentration of primary antibody. |
| Non-specific | Protein forms multimers | Boil sample longer in Laemmli buffer to fully reduce disulfide bonds. |
| Non-specific | Cells passaged too many times | Use the original, non-passaged cell line. |
| Non-specific | Antibodies not purified | Use affinity-purified antibodies. |
| Non-specific | Bands are non-specific | Use blocking peptides to distinguish specific vs non-specific bands. |
| Non-specific | Post-translational modification | Multiple modified forms (acetylation, methylation, phosphorylation) — check literature. |
| Non-specific | Ionic interactions | Increase salt in incubation buffers; add stronger detergent to washes. |
| Low MW | Samples digested/degraded | Use fresh sample and lysis buffer with proteinase inhibitors. |
| Low MW | Primary detecting splice variants | Identify splice variants; try a different primary. |
| Low MW | Primary binding a similar epitope | Run a negative control to detect cross-reacting proteins. |
| High MW | Protein aggregation | Decrease protein concentration; prepare new sample with fresh loading buffer. |
| High MW | Incomplete denaturation | Denature protein with urea. |
| High MW | Proteins are glycosylated | Use enzymatic treatment to strip post-translational modifications. |
| Poor transfer | Membrane choice | Choose PVDF/nitrocellulose to match the target molecular weight. |
| Poor transfer | Dry membrane | Do not let the membrane or filter paper dry out. |
| Poor transfer | Incomplete protein resolution | Use an optimal gel percentage for the protein of interest. |
| Poor transfer | Incorrect sample preparation | Sample must contain DTT or β-mercaptoethanol and be heated before loading. |
| Poor transfer | Incorrect membrane assembly | Orient PVDF/NC membranes on the anode (+) side of the gel. |
| Poor transfer | Incorrect transfer time | Increase transfer time to improve transfer of large proteins. |
| Poor transfer | Insufficient power supply | Membranes can be placed either side of the gel if the supply is mis-connected. |
| High background | Non-specific antibody binding | Use the most specific primary antibody available. |
| High background | Insufficient blocking | Optimise blocking-time duration. |
| High background | Incomplete blocking | Optimise blocking buffer; increase protein in the blocking agent. |
| High background | Incompatible blocking agent | Compare different blocking buffers. |
| High background | Incorrect membrane choice | Nitrocellulose generally gives less background than PVDF. |
| High background | Film overexposed | Reduce the exposure time. |
| High background | Secondary binding blocking reagent | Add Tween 20 to washing and incubation buffers. |
| High background | Incubation temperature too high | Incubate at 4°C; keep samples on ice. |
| High background | Excessive incubation | Reduce incubation time. |
| High background | Too much substrate | Dilute substrate; reduce substrate incubation time. |
| High background | Protein overloaded | Dilute the sample or reduce load. |
| High background | Milk contains target antigen | Substitute with 3% BSA. |
| High background | Suboptimal antibody concentration | Optimise antibody concentration. |
| High background | Insufficient washing | Increase number of washes; increase Tween 20 in wash buffer. |
| High background | Blot dried out | Cover the membrane in buffer during incubation. |
| High background | Antibody cross-reactivity | Reduce secondary concentration; use a different blocking agent. |
| High background | Contamination of membranes, solutions or trays | Wear clean gloves; handle membranes with forceps; use clean glassware and distilled water; run a cleaning protocol. |
| Speckled | Blocking reagent clumped | Filter the blocking agent to remove clumps. |
| Speckled | Contamination of gel or reagents | Use fresh, sterile buffer. |
| Speckled | Exposure time too long | Reduce the exposure time. |
| Speckled | Insufficient solution during wash/incubation | Fully immerse the membrane during incubations and washes. |
| Speckled | Contamination of equipment | Wash electrophoresis equipment and membrane thoroughly. |
| Speckled | Uneven agitation | Use a rocker/shaker for uniform agitation. |
| Speckled | Secondary antibody aggregation | Increase secondary dilution; spin down aggregates. |
| Speckled | Membrane dried unevenly | Thoroughly wet the membrane and keep it from drying out. |
| White spots | Air bubbles against the membrane | Remove air bubbles between membrane and gel during transfer. |
| Bands low | Gel ran too long | Run the gel for a shorter period. |
| Bands low | Too little acrylamide | Run low-MW proteins in higher-percentage gels. |
| Not fully run | Gel not run long enough | Run the gel for a longer period. |
| Not fully run | Too much acrylamide | Run high-MW proteins in lower-percentage gels. |
| Distorted | Voltage too high during migration | Check the protocol for the recommended voltage. |
| Distorted | Gel too hot during migration | Run the gel at 4°C, on ice or in a cold room. |
| Uneven | Gel polymerised unevenly | Check gel recipe/TEMED; ensure the gel is fully covered in buffer when setting. |
| Uneven | Salt varies between wells | Ensure similar salt concentration across samples. |
| Diffuse/streak | Excessive protein on gel | Reduce the amount of protein loaded. |
| Diffuse/streak | Membrane slipped during transfer | Avoid moving the gel or membrane during transfer. |
| Diffuse/streak | Incomplete membrane-gel contact | Use thicker filter paper; squeeze out excess buffer and air bubbles. |
| Blurry | Electrophoresis voltage too high | Run the gel longer at a lower voltage. |
| Blurry | Air bubbles between membrane and gel | Remove bubbles by rolling with a sterile glass rod. |
| Blurry | Incorrect loading buffer | Prepare fresh loading buffer. |
| White bands (ECL) | Antibody concentration too high | Dilute the antibody to the optimal concentration. |
| White bands (ECL) | Excessive signal generated | Reduce the concentration of protein or antibody. |
| White bands (ECL) | Overexposure during visualisation | Decrease exposure time. |
| White bands (ECL) | Blot moved during transfer | Avoid moving membrane or gel during transfer. |
| White bands (ECL) | Loading sample too concentrated | Reduce the amount of sample loaded. |
All Western blot buffers & recipes
Every essential buffer in one place — lysis, gel, running, transfer, blocking, wash and stripping.
RIPA buffer
- 50 mM Tris-HCl pH 7.4
- 50 mM NaCl
- 2 mM EDTA
- 1% SDS
- + fresh protease inhibitors
NP-40 buffer
- 150 mM NaCl
- 1.0% NP-40 (or Triton X-100)
- 50 mM Tris pH 8.0
Tris-HCl buffer
- 20 mM Tris-HCl pH 7
Tris-Triton buffer
- 10 mM Tris pH 7.4
- 100 mM NaCl
- 1 mM EDTA
- 1 mM EGTA
- 1% Triton X-100
- 10% glycerol
- 1% SDS
- 5% deoxycholate
Laemmli buffer
- 4% SDS
- 10% 2-mercaptoethanol
- 20% glycerol
- 0.004% bromophenol blue
- 0.125 M Tris-HCl pH 6.8
Loading buffer (2×)
- 100 mM Tris-HCl pH 6.8
- 200 mM DTT
- 4% SDS
- 0.2% bromophenol blue
- 20% glycerol
- (add DTT fresh from 1 M stock)
10% resolving gel
- H₂O 5.9 mL
- 30% acrylamide-bis 5 mL
- 1.5 M Tris pH 8.8 3.8 mL
- 10% SDS 150 µl
- 10% APS 150 µl
- TEMED 6 µl
Stacking gel
- H₂O 2.7 mL
- acrylamide-bis 670 µl
- Tris pH 6.8 500 µl
- 10% SDS 40 µl
- 10% APS 40 µl
- TEMED 3 µl
Running buffer
- 15.1 g Trizma
- 94 g glycine
- 50 mL 10% w/v SDS
- dH₂O to 1 L
Transfer buffer
- 8 g Trizma base
- 9 g glycine
- 3.7 g SDS
- 200 mL methanol
- dH₂O to 1 L
Blocking buffer
- 5% marvel in TBST
Wash buffer (TBS-T)
- 20 mM Tris pH 7.5
- 150 mM NaCl
- 0.1% Tween-20
Mild stripping buffer
- 15 g glycine
- 1 g SDS
- 10 mL Tween-20
- 800 mL dH₂O, pH 2.2
- bring to 1 L
Harsh stripping buffer
- 62.5 mM Tris-HCl pH 7.8
- 100 mM β-mercaptoethanol
- 2% (w/v) SDS
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