1. A novice's guide-WESTERN BLOTTING
Prepared by: 
Dewan Md. Sumsuzzman,
Lab: Biological clock & aging.
Department of Rehabilitation Science, Inje University, South Korea.

2. INTRODUCTION Why western blotting? Protein detection
Protein quantification
What is WB?
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. 
Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis. The blot is a membrane, almost always of nitrocellulose or PVDF (polyvinylidene fluoride).

3. STEPS OF WESTERN BLOTTING
Sample preparation
Gel electrophoresis
Transfer from gel to membrane
Immunoblotting.

4. Sample preparation Lysis buffers Protease and phosphatase inhibitors
Preparation of lysate from cell culture
Preparation of lysate from tissues
Determination of protein concentration
Preparation of samples for loading into gels

5. SAMPLE PREPARATION Lysis buffer
The first step in sample preparation is isolating proteins from their source.
Usually, Protein is isolated from cells and tissues via lysis. Lysis breaks down the cell membrane to separate proteins from the non-soluble parts of the cells. 
There are various lysis buffers that can be used for sample preparation in WB. 
Lysis buffer vary in the strength of their detergents to release soluble proteins.
Example: SDS, Triton X-100.
Selection of optimal lysis buffer depends on- Location of the protein of interest- For obtaining high purity and yield.

6. Membrane-bound proteins
SAMPLE PREPARATION
Subcellular location
Recommended buffer
Whole cell lysate
NP-40
Nucleus
RIPA
Mitochondria
Cytoplasm
Tris-Hcl
Membrane-bound proteins

7. SAMPLE PREPARATION NP-40 RIPA Tris-Hcl 150 mM NaCl 20 mM Tris-Hcl
1% NP-40 Or Triton X-100
PH-7.5
50 mM Tris 
0.5% Sodium Deoxycholate
PH-8.0
0.1% SDS
50 mM Tris

8. SAMPLE PREPARATION Protease and phosphatase inhibitors
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down tremendously if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer.

9. SAMPLE PREPARATION Inhibitor Protease/phosphatase Inhibited
Final concentration
in lysis buffer
Stock (store at -20oC)
Aprotinin
Trypsin, Chymotrypsin,
Plasmin
2 μg/ml
D i l u t e in w a t e r, 10 mg/ml. Do not reuse thawed aliquots
Leupeptin
Lysosomal
5-10 μg/ml
D i lu t e i n w a t e r . D o not re-use once
defrosted.
Pepstatin A
Aspartic proteases
1 μg/ml
D i l u t e in m e t h anol, 1mM.
PMSF
Serine, Cysteine proteases
1 mM
Dilute in ethanol. You can re-use the
same aliquot.
EDTA
Metalloproteases that require
Mg++ and Mn++
5 mM
Dilute in dH2O, 0.5M. Adjust pH to 8.0.
EGTA
Ca++
Na Fluoride
Serine/Threonine
phosphatases
5-10 mM
Dilute in water. Do not re-use once Na
Orthovanadate
Tyrosine phosphatases

10. SAMPLE PREPARATION NO. STORE SOLUTION FUNCTION STOKE 10 ml 1 RT
50 mM Tris (pH-7.4)
PH buffer
1M (100mM)
500 µL 2
150 mM NaCl
Saline buffer
1M (300mM)
1.5 mL 3
0.1% SDS
Anionic detergent, Protein & lipid solubilizing reagent
10%
100 µL 4
1% NP-40 (light protected)
Non-ionic detergent, Solubilizing membrane protein
10% in water (Boiled 1ml/9ml)
1mL 5
0.5% Sodium Deoxycholate
Ionic detergent, Nuclear membrane disruption
10% in water (0.1g/1mL) 6
-20
1mM Na Orthovanadate
Inhibitor of all tyrosine phosphatases
200 mM
50 µL 7
5 mM Na Fluoride
Inhibitor of Serine/Threonine phosphatases
500 mM (0.021g/1mL) 8
Protease inhibitor cocktail
Serine, Cysteine, Metallo-proteases inhibitor
1 Tablet 9
1 mM PMSF
Serine proteases inhibitor, Cysteine proteases inhibitor, Aspartic proteases
200 mM in isopropanol ( g/1mL) 10
Autoclaved DW
6.2 mL

11. SAMPLE PREPARATION Preparation of lysate from cell culture
1. Place the cell culture dish in ice and wash the cells with ice-cold PBS.
2. Drain the PBS, then add ice-cold lysis buffer (1 ml per 107 cells/100mm dish/150 cm2 flask; 0.5ml per 5x106 cells/60mm dish/75cm2 flask).
3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microfuge tube.
4. Maintain constant agitation for 30 minutes at 4°C.
5. Centrifuge in a microcentrifuge at 4°C.
(You may have to vary the centrifugation force and time depending on the cell type; a guideline is 20 minutes at 2,000 rpm but this must be determined by the end-user (e.g. leukocytes need a very light centrifugation).
6. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.

12. SAMPLE PREPARATION Preparation of lysate from tissues
1. Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.
2. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to “snap freeze”. Store samples at -80°C for later use or keep on ice for immediate homogenization.
3. Centrifuge for 20 min at rpm at 4°C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.

13. SAMPLE PREPARATION Determination of protein concentration
It is important to determine the protein concentration of each lysate to ensure equal loading of the SDS-PAGE. 
Protein concentration can be determined by performing a standard Bradford, Lowry or BCA assay.
Once you have determined the concentration of each sample, you can freeze them at -20°C or -80°C for later use.

14. SAMPLE PREPARATION Loading buffer
2x Laemmli Buffer (Loading buffer pH-6.8)
Preparation of samples for loading into gels
The epitope usually resides within the 3D conformation of the protein. Thus it is necessary to unfold or denature the protein to enable access to the antibody.
Denaturing is performed by briefly boiling the sample in a loading buffer containing SDS.
The most common loading buffer is 2X Laemmli buffer. It can also be made at other concentration such as 4X or 6X, which may helpful if loading larger volumes of lysates with low protein concentration.
REAGENT 2X
2X *10 mL
20 mL
Function
1M Tris-C(pH-6.8)
100 mM(0.1M)
1mL
2mL
100% Glycerol
20%
4mL
Increased sample density
10 SDS 4%
4 mL
8mL
Denatured protein
Β-Mercaptoethanol 5%
500 µL
1 mL
Reducing agent
4 Bromophenol blue
2 mL 480 µL
Anionic dye to visualize protein migration.

15. Gel electrophoresis Principle Steps
After sample preparation and loading buffer addition, sample must be loaded onto a gel. 
Electrophoresis is performed with a negative pole on the well end of the gel and a positive pole on the opposite end of the gel. 
The negative charged SDS bound to protein causes migration of protein complexes towards the positive pole during electrophoresis and protein can be separated by size.
In general, the larger the protein, the slower it moves through the gel.
 Preparation of PAGE gel
Positive control
Molecular wt. Marker
Loading sample and running the gel
Use of loading control

16. GEL ELECTROPHORESIS Preparation of PAGE gel Positive control
Selection of acrylamide % 
A positive control lysate is used to demonstrate that the protocol is efficient and correct and that the antibody recognizes the target protein which may not be present in the experimental samples.
Protein size (KDa)
Gel percentage (%)
10-43 15
12-60 12
20-80 10
36-94
7.5
57-212 5

17. Gel electrophoresis Loading control Sample type Molecular weight (kDa)
Caution
Beta Actin
Whole Cell / cytoplasmic 43
Not suitable for skeletal muscle samples. Changes in cell-growth conditions and interactions with extracellular matrix components may alter actin protein synthesis.
GAPDH
30-40
Some physiological factors, such as hypoxia and diabetes, increase GAPDH expression in certain cell types.
Tubulin 55
Tubulin expression may vary according to resistance to antimicrobial and antimitotic drugs.
VDCA1/Porin
Mitochondrial 31
COXIV 16
Many proteins run at the same 16 kDa size as COXIV.
Lamin B1
Nuclear 66
Not suitable for samples where the nuclear envelope is removed.
TATA binding
protein TBP 38
Not suitable for samples where DNA is removed

18. GEL ELECTROPHORESIS

19. GEL ELECTROPHORESIS Running gel-10ml Stacking gel-55%
7.5%
10%
12%
15%
30 Acrylamide sol.
2ml
2.7ml
3.3ml
4ml
5ml
1.5 M Tris-Cl
(pH-8.8)
2.5ml
2.5
10 SDS
100 µL
100µL
100
10 APS
TEMED
8µL
6µL
4µL 4
D.W
5.3ML
4.6ML
4ML
3.3
2.3ML
5% stacking gel
2ml
4ml
6ml
8ml
30 Acrylamide sol.
330µl
670µl
1ml
1.3ml
1.5 M Tris-Cl
(pH-6.8)
250µl
500µl
750µl
10 SDS
20µl
40µl
60µl
80µl
10 APS
TEMED
2µl
4µl
6µl
8µl
D.W
1.4ml
2.7ml
4.1ml
5.5ml

20. GEL ELECTROPHORESIS

21. Transfer protein from gel to membrane
After Gel-electrophoresis, protein must be transferred from the gel onto a suitable membrane for subsequent immunoassay steps. This is performed by passing an electrical current across the gel to the membrane.
The membrane can be either PVDF or nitrocellulose.
PVDF is better for low molecular wt. Proteins.
For protein less than 30 KDa, the pore size of 0.2 µm PVDF is recommended over the  µm pore size.
Additionally, PVDF have a higher binding capacity (  µg/cm2) than nitrocellulose (80-100µg/cm2).

22. Transfer protein from gel to membrane
1x transfer buffer (pH-8.3), 1L
10x transfer buffer
100 ml
MetOH
200 ML
D.W
700 ML
10x transfer buffer (pH-8.3),1L
Tris (250 mM)
30.28 G
Glycine (1920 mM)
140 G
Fig: Sandwich assembly

23. TRANSFER PROTEIN FROM GEL TO MEMBRANE
Prepare membrane by wetting it in methanol for 30 sec and then soaking it briefly in distilled water followed by 1x transfer buffer.
Soak filter papers and sponges in the transfer buffer for 10 mins prior to assembly of the transfer "SANDWICH"
After electrophoresis, remove the gel from the electrophoresis apparatus and equilibrate it by soaking in transfer buffer 10 mins.
Prepare the sandwich according to the previous slide illustration. Sequentially assembly the layers of the sandwich gently remove any air with roller. 
Bubbles between the gel and membrane will inhibit the transfer of protein to the membrane.
Place the sandwich into the transfer cassette and perform semi-dry or wet transfer according to the manufacturer instructions of the blotting apparatus.
Semi-dry: Faster, Suitable for >100 Kda protein; Wet transfer: <30 KDa protein.

24. Immunoblotting Blocking:
The first step in immunoblotting is to rinse and block the membrane with non-specific protein, such as milk or BSA. 
The purpose of blocking step is to bind non-specific protein to the surface of the membrane where sample protein is not already present. This prevents antibody from binding non-specifically to the membrane, which gives rise to a high background signal.
Often the antibody information datasheet will recommend non-specific protein either milk or BSA.
Milk should be avoided, as it contains a high amount of phosphorylated proteins.

25. Immunoblotting 1x TBST, 1L Blocking buffer TBST-500 ML Skim milk-25 g
D.W
900 ml
Tween-20
1 ml
TBST-500 ML
Skim milk-25 g
BSA-1%

26. IMMUNOBLOTTING Incubation with primary antibody
Incubation of secondary antibody
Incubation buffer: Dilute the antibody in TBST according to (1:100-1:3000) range of dilutions if the datasheet does not have recommended.
Incubation time: Few hours to overnight. And it depends upon the binding affinity of the antibody for the protein and the abundance of protein.
Incubation temp: Preferably cold. If incubating in blocking buffer overnight, it is vital important to incubate at 4°C or contaminate will occur and thus destruction of the protein. Agitation of the antibody is recommended to enable adequate homogenous covering of the membrane and prevent uneven binding.
Wash the membrane several times in TBST while agitating, 5 mins or more per wash, to remove residual primary antibody.
Incubation buffer & dilution: If the datasheet does not have recommended dilution, try a range of dilutions (1:1000-1:20,000) and optimize the dilution according to the results. 
Incubation time & temp: 1-2 hours, room temperature, with agitation.
Note: HRP-conjugated secondary antibody is always recommended. ALP- conjugated secondary antibody are not sensitive enough.

27. Fig: Detection mechanism
X-ray films
Detection kits
Digital image
Fig: Detection mechanism

28. Thank you