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

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).

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

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

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.

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

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

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.

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

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 (0.03484g/1mL) 10 Autoclaved DW 6.2 mL

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.

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 12000 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.

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.

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.

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

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

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

GEL ELECTROPHORESIS

GEL ELECTROPHORESIS Running gel-10ml Stacking gel-5 5% 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

GEL ELECTROPHORESIS

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 0.45 µm pore size. Additionally, PVDF have a higher binding capacity (150-160 µg/cm2) than nitrocellulose (80-100µg/cm2).

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

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.

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.

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%

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.

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

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