WESTERN BLOTTING ADYA.pptx

BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.

1. बाबासाहेब भीमराव अम्बेडकर विश्वविद्यालय Babasaheb Bhimrao Ambedkar University (A Central University) Accredited 'A' Grade by NAAC 2015 (In First Cycle) ISO 14001:2015 WESTERN BLOTTING Presented By :- AADYA RAJ PANDEY 1 st Year M.Pharm (Pharmacology SUBMITTED TO: PROF: GAURAV KAITHWAS SIR

2. Table of content Principle Reagents required Procedure Tissue Preparation (preparation of sample lysate): Gel Electrophoresis: Transfer Immunoblotting Detection Steps: Detection can be done by other methods such as: Colorimetric detection Radioactive detection Fluorescent detection Uses

3. Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures. It is the technique för transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis . What is a blot?

4. IMMUNO BLOTTING TECHNIQUES Immunoblotting techniques use antibodies to identify target proteins . They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions. The Southern blot is used for transferring DNA , . The Northern blot for RNA The western blot for PROTEIN . The Eastern blot for PROTEIN , post-translational modifications (PTMS) . ( Sir Edwin Mellor Southern)

5. IMMUNO BLOTTING TECHNIQUES Used to detect RNA Used to detect protein Used to detect protein Used to detect DNA

6. WESTERN BLOTTING Principle: Western blotting technique is used for identification of particular protein from the mixture of protein. In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.

7. Western blotting procedure:

8. PROCUDURE: 1. Extraction of protein 2. Gel electrophoresis: SDS PAGE 3. Blotting: electrical or capillary blotting 4. Blocking: BSA 5. Treatment with primary antibody 6. Treatment with secondary antibody( enzyme labelled anti Ab) 7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color. separation transfer detection

9. Key solutions and reagents : 1.Lysis buffer: Radioimmunoprecipitation Assay buffer. 50 mM Tris-HCl, pH 8.0 150 mM NaCl 1% Nonidet P-40 (NP-40) or 0.1% Triton X-100 (lyase cell membrane only) 0.5% sodium deoxycholate( disrupt protein interaction for liposome and membrane protein isolation.) 0.1% sodium dodecyl sulphate (SDS) 1 mM NaF Protease inhibitors tablet (Roche)(inactivate endogenous phosphates and protect protein phosphorylation) .

10. Preparation of gel: Chemical copolymerization of acrylamide monomers with cross linking reagent N-N’-methylene bis-acrylamide results in a clear transparent gel. It is a free radical catalysis reaction initiated by ammonium per sulfate and TEMED. TEMED catalyses the persulfate ion to start free radical generation.

11. 2.Loading buffer: 2x Laemli buffer solution with high density , which facilitate loading of protein , DNA, RNA, containing solutions into wells of agarose gel. Chemical constituent Concentration uses SDS ( Sodium dodecyl sulfate) 4% Anionic Detergent 2-mercaptoethanol 10% breaks non covalent bond glycerol 20% Gives density and high resolution bromophenol 0.004% blue tracing dye Tris HcL 0.125 M Contribute as zwitter ion

12. 3.Running buffer & transfer buffer Running buffer: Tris/Glycine/SDS 25 mM Tris- 3 190 mM glycine 0.1% SDS Transfer buffer : Tris /Glycine /SDS 25 mM Tris 190 mM glycine 20% methanol For proteins larger than 80 KD, we recommend that SDS be included at a final concentration of 0.1%. Provides conductive media for tranfer Main purpose is to create an electric field that allows for movement of proteins through gel during electrophoresis .

13. 4.Ponceau S staining buffer Ponceau S staining buffer0.2% (w/v) Ponceau S5% glacial acetic acid✔ 5. Tris-buffered saline with Tween 20 (TBST) TBST buffer20 mM Tris, pH 7.5 150 mM NaCl 0.1% Tween 20 6.Blocking Buffer : 3% bovine serum albumin (BSA) in TBST 7. Stripping buffer: 20 ml 10% SDS 12.5 ml 0.5 M Tris HCl, pH 6.8 67.5 ml ultrapure water 0.8 ml 2-mercaptoethanol

14. STEP 1: Extraction of protein Cell lysate is most common sample for western blotting. Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also known as tissue preparation. Cell Lysis : Chemical Lysis: Cells are treated with detergents or other chemical agents to disrupt cell membranes and release proteins. Mechanical Lysis : Cells are physically broken open using methods like homogenization or sonication. Centrifugation : After cell or tissue lysis, centrifugation is often used to separate cell debris, organelles, and other contaminants from the protein-containing supernatant. Precipitation : Protein precipitation methods, such as ammonium sulfate or acetone precipitation, are used to concentrate proteins by causing them to come out of solution. To prevent denaturing of protein protease inhibitor is used. The concentration of protein is determined by spectroscopy. . When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing glycerol which helps to sink the sample in well. Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.

15. Continue: Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout). Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes. Centrifuge at 1500 RPM for 5 minutes and discard the supernatant. Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail). Incubate for 30 minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C. Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C.

16. Sample preparation :

17. Step II: Gel electrophoresis The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis.. The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of these all.. The small size protein moves faster than large size protein. Protein are negatively charged, so they move toward positive (anode) pole as electric current is applied.

18. Step III: Blotting The nitrocellulose membrane is placed on the gel. The separated protein from gel get transferred to nitrocellulose paper by capillary action. This type of blotting is time consuming and may take 1-2 days For fast and more efficient transfer of desired protein from the gel to nitrocellulose paper electro-blotting can be used. In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.

19. Step IV: Blocking Blocking is very important step in western blotting. Antibodies are also protein so they are likely to bind the nitrocellulose paper. So before adding the primary antibody the membrane is non- specifically saturated or masked by using casein or Bovine serum albumin (BSA). Step V: Treatment with Primary Antibody The primary antibody (1 Ab) is specific to desired protein so it form Ag-Ab complex

20. Step VI: Treatment with secondary antibody The secondary antibody is enzyme labelled. For eg. alkalinephosphatase or Horseradish peroxidase (HRP) is labelled with secondary antibody. Secondary antibody (2" Ab) is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab complex.

21. Step VII: Treatment with suitable substrate . To visualize the enzyme action, the reaction mixture is incubated with specific substrate.. The enzyme convert the substrate to give visible colored product, so band of color can be visualized in the membrane.. Western blotting is also a quantitative test to determine the amount of protein in sample.

22. 7. Washing . Removes unbounded antibodies from the membrane. Commonly used buffer: a dilute solution of tween-20 in TBS or PBS buffer. 8. Protein Detection • Alkaline phosphates (AP) and horse radish peroxidase (HRP) are widely used. Four types OF analysis techniques: Chromogenic detection Chemiluminescence detection Fluorescent detection radioactive detection

23. 9. Analysis and Imaging Detection of signals using X-ray film, scanners or CCD. Benchmarking with marker protein to estimate the molecular weight of the protein. Verification can be done through qualitative and quantitative analysis to show the presence and absence of specific proteins of interests.

24. Applications Detection of particular protein from a mixture of proteins. Size and amount estimation of proteins in the mixture.. Western blotting is a widely used laboratory technique in cancer research that helps detect specific proteins within a cell or tissue sample. It has several applications in cancer detection and treatment, including the identification of cancer-specific proteins and monitoring treatment response. 1. *p53 (TP53) Gene *: Mutations in the TP53 gene are common in various cancers. Western blotting can be used to detect the presence and activity of p53 protein in tumor samples. Loss of p53 function is associated with uncontrolled cell growth and is a hallmark of many cancers. 2. *HER2 (ERBB2) Gene*: HER2-positive breast cancer is characterized by overexpression of the HER2 protein. Western blotting can confirm the overexpression of HER2 protein, which guides treatment decisions involving targeted therapies like Herceptin.

25. Continue.. 3. * BRCA1 and BRCA2 Genes *: Mutations in these genes increase the risk of breast and ovarian cancer. Western blotting can be used to study the protein products of these genes and their involvement in DNA repair pathways. 4. * BRAF Gene *: Mutations in the BRAF gene are found in melanoma and certain other cancers. Western blotting can be employed to assess the activation status of the BRAF protein and guide the use of targeted therapies like BRAF inhibitors. 5. * EGFR Gene *: Overexpression or mutations in the Epidermal Growth Factor Receptor (EGFR) gene are associated with several cancer types, including lung cancer. Western blotting can be used to determine EGFR protein levels and activation status, aiding in treatment decisions. 6. * AKT (Protein Kinase B) Gene *: The AKT pathway is frequently dysregulated in cancer. Western blotting can be used to assess the phosphorylation status of AKT and downstream signaling proteins, helping to identify potential therapeutic targets.

26. continue: 1. * Protein Expression Analysis: used to assess the expression levels of specific proteins in cancer cells or tissues. Researchers can compare protein profiles between normal and cancerous tissues, helping to identify potential biomarkers associated with cancer. 2. * Diagnostic Biomarkers:* Specific proteins may serve as biomarkers for different types of cancer. Western blotting can help validate the presence or absence of these biomarkers in patient samples, aiding in cancer diagnosis and classification. 3. Drug Target Identification 4 . *Research into Resistance Mechanisms:* By tracking changes in protein expression over time, researchers can uncover the molecular mechanisms behind resistance and explore strategies to overcome it.

27. Disadvantages Requires specific primary antibodies to perform test on desired protein of interest. Challenging and hence requires well trained staffs. Poorer results as antibodies may revel off-target bindings. Detecting and imaging the results can be expensive as equipment cost is high.

28. Conclusion Western blotting is crucial for understanding the expression, activation, and post-translational modifications of these and many other proteins involved in cancer development and progression. use of Western blotting to study the effects of potential drug candidates on these proteins, which can inform the development of targeted cancer therapies. Additionally, Western blotting can be employed to assess the expression of specific proteins as biomarkers for cancer diagnosis, prognosis, and treatment response.