Western Blot Sample Protocol

Western Blot Sample Protocol – Introduction

Western blotting is a method used to detect and analyse specific protein molecules in a sample using antibodies. Western blots can be used to determine the size of your protein of interest and measure the amount of protein expressed and what post-translational modifications your protein of interest may have undergone following treatment with a drug, environmental or genetic changes.

Following protein extraction using either RIPA buffer or cell lysis buffer protein mixture samples can be loaded into wells of a PAGE gel, proteins are then separated by gel electrophoresis. Following electrophoresis proteins are transferred to a nitrocellulose or PVDF membrane via application of an electrical current. Once the proteins are on the membrane they can then be probed with specific antibodies for your protein of choice and visualized using secondary antibodies and detection regents.  In this guide you will find solutions and regent recipes for Western blots in addition to a sample protocol which can be modified for your own use.

Solutions and Reagents needed

  • Lysis Buffer (RIPA Buffer)
  • Loading Buffer
  • Running Buffer
  • Transfer Buffer
  • TBS-T
  • Blocking Buffer
  • Stripping Buffer

Procedure

Sample Preparation

  1. Lyse cells in RIPA buffer (1% v/v NP40 in 20 mM Tris-HCL ph 7.4, 5 mM NaF, 5 mM sodium pyrophosphate, 5 mM EDTA) with freshly added proteinase inhibitors (Leupeptin, PMSF, Sodium Ortovanadate) to decrease protein degradation. For full protocol click here.
  2. Collect the cell lysate and determine protein concentration using BCA or Bradford assay.
  3. Take 20ug of sample and add an equal volume of 2x laemmli buffer. Laemmli buffer contains beta-2-mercaptoethanol which acts to reduce disulfide bonds and in turn denatures the protein.
  4. Denature samples on a heating block for 5 minutes at 95°C.

Sample Separation by SDS-PAGE Gel electrophoresis

  1. Separate proteins by SDS-PAGE on an acrylamide gel. The percentage of acrylamide used will depend on the molecular size of your protein of interest. In this sample protocol a 10% gel is described. In the table below you will find a guide to acrylamide concentration and best practice separation ranges for proteins.
Acrylamide concentration % Linear Range of Seperation (KD)
5.0 57-211
7.5 36-94
10 16-68
15 12-43

Table 1. Percentage acrylamide gel guide.

  1. Poor the resolving gel between the plates and let the gel set for 1 hr.
  2. Level the gel with isopronol. For this take 300 uL of Isopropanol and poor carefully across the top of the gel.
  3. Once your gel has set you can remove the Isopropanol by puring a gentle flow of water over the gel under the tap.
  4. Add the stacking gel and carefully place your comb into the gel, avoiding air bubbles. Allow gel to set for 1 hr.
Reagent Volume
H20 5.9 ml
30% Acrylamide-Bis 5 ml
1.5M Tris pH 8.8 3.8 ml
10% SDS 150 µl
10% APS 150 µl
Temed 6 µl

Table 2: 10% Resolving Gel Recipe

Reagent Volume
H20 2.7 ml
Acrylamide-Bis 670 µl
Tris pH 6.8 500 µl
10% SDS 40 µl
10% APS 40 µl
Temed 3 µl

Table 3: Stacking Gel Recipe

  1. Place the plate in the gel electrophoresis rig and add 400ml of 1x SDS-PAGE running buffer (15.1 g TRIZMA, 94 g glycine,50 ml of 10% w/v SDS,dH20 to 1 litre), first to the inside of the chamber and let over flow out to ensure no leakage.
  2. Remove the combs and wash out the wells with running buffer if necessary.
  3. Add 8 µl of unstained molecular weight ladder and 20 µl of the protein samples of interest to the selected well.
  4. Run the gels at 25mA per gel for 1 hr 15 min.

Western Blot Transfer

  1. Remove the glass plates from the electrophoresis apparatus and place them on a paper towel. Using a spatula, pry the plates apart.
  2. Mark the orientation of the gel by cutting a corner from the bottom of the gel.
  3. Incubate the gel Transfer Buffer for approximately 10 min to remove detergent.
  4. Cut the membrane and filter paper to the dimensions of the gel.
  5. Equilibrate Whatman filter paper in ice cold transfer buffer (5.8g Trizma base, 2.9g glycine, 0.37g SDS powder, 200ml methanol dH20 to 1L) for 15 min.
  6. Activate the Polyvinylidene difluoride (PVDF) membrane in methanol (100%) for 20 sec, then wash with ddH 2O and equilibrate in transfer buffer for 15 min.
  7. Place the gel on three sheets of Whatman filter paper (3mM) followed by the activated PVDF membrane.
  8. Add Another three sheets of filter on top,
  9. Place in a transfer cassette with the gel closest to the black side.
  10. Remove any air bubbles, which may have formed. Using a glass tube to gently roll air bubbles out.
  11. Add the last fiber pad. Ensure the sandwhich is thoroughly covered in transfer buffer
  12. Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the white latch.
  13. The protein within the gel was transferred to PVDF membranes using a tank transfer system for 60 min at 60 V and 180 mA.
  14. Block the PVDF with the transferred protein in 5% marvel in TBS-Tween (1X TBS with 0.1% v/v tween-20) for 30 minutes at room temperature on a shaker.
  15. Wash with TBS-T (Western blot buffer) 5 times for 5 minutes at room temperature before incubation with the primary antibody.
  16. Add the primary and incubate in the cold room. The duration may vary from antibody to antibody and can in some instances be left overnight.
  17. Wash with x5 time for 5 min with TBS-T
  18. Incubate the membrane with the secondary antibody containing a flurescent tag eg HRP
  19. Repeat step 17
  20. Viusliase the protein bands on the PVDF membrane using enhanced chemiluminescence (ECL) reagent or exposing the membrane to X-ray film in the dark room
  21. To ensure that each well is equally loaded the blot can be probed for Tubulin or a relevant housekeeping gene.

Stripping and re-probing of Western Blots

  1. Incubate the PVDF Membrane with stripping buffer (62.5 mM Tris-HCL,pH 7.8, 100 mM -mercaptoethanol, 2% (w/v) SDS) for 30 min at 50 °C.
  2. Wash the membrane 2 X 5 min, 1 X 10 min, 2 X 5 min at room temperature with agitation on a platform rocker.
  3. Block by incubation with gentle agitation in blocking solution for 1 h at room temperature.
  4. Washed 2 X 5 min, followed by incubation with the appropriate primary and secondary antibody.

Western Blot Solutions and Reagents Recipes

Loading Buffer Recipe

  • 2X Loading buffer:
  • 100 mM Tris-HCL (pH6.8)
  • 200 mM DTT
  • 4% SDS
  • 0.2% Bromophenol blue
  • 20% glycerol
  • (DTT should be added just before the buffer is used, from 1M stock)

Running Buffer Recipe

  • 15.1 g TRIZMA,
  • 94 g glycine
  • 50 ml of 10% w/v SDS,
  • dH20 to 1 litre

Transfer Buffer Recipe

  • 5.8g Trizma base
  • 2.9g glycine
  • 0.37g SDS powder
  • 200ml methanol
  • dH20 to 1L

TBS-T Recipe

  • 20 mM Tris, pH 7.5
  • 150 mM NaCl
  • 0.01% Tween 20

Blocking Buffer

  • 5% Marvel in TBST

Stripping Buffer

  • 62.5 mM Tris-HCL,pH 7.8,
  • 100 mM -mercaptoethanol,
  • 2% (w/v) SDS

Helpful Hints

  • As a Western blot can take a lot of time and effort – always start with a good quality sample. Poor protein extraction and preparation techniques will result in a bad Western blot and more importantly wasted time.
  • Making your gels can be a pain, especially when you return to find they haven’t set – probably because you left a component. You can check if your gel has set by tilting the rig, if its still liquid your best and most time efficient option is to start again. If you can afford it commercial solutions are available in form of pre-cast gels.
  • During the transfer stage its easy to forget about your gel and let it dry out. This will result in a bad blot. To prevent this following electrophoresis always keep your gel in some transfer buffer. This will give you time to cut and prepare your Whatman filter paper and PVDF membrane. Alternatively you prepare these in bulk and store away for when needed,
  • Dont underestimate your shaker settings. Adjust the shaker to the right speed (not too fast or too slow). If the shaker is too fast the antibody wont bind correctly, if its too slow non-uniform binding can occur.
  • If you reuse your antibodies always mark on the side how many times youve reused it. It may explain why blots that were working brilliantly and suddenly acting up.
  • Make sure you use the right loading control it might not always be GAPDH. Please see the list below for some suggested loading controls depending on sample type.
Sample type Protein MW (kDa)
whole cell / cytoplasmic proteins
  beta actin 43
  alpha actin 43
  GAPDH 30-40
  beta tubulin 55
  alpha tubulin 55
(high molecular weight) vinculin 116
Mitochondria
  VDCA1/porin 31
  cytochrome C oxidase 16
Nuclear proteins
  lamin B1 66
  TATA binding protein TBP 38
  PCNA 29
  histone H1  
  histone H3  
Plant tissue
  LHCP  
  APX3  
Serum sample
  transferrin 77
Muscle sample
  SDHA [7] 73
Yeast sample
  phosphoglycerate kinase