Western Blot .pptx

A Western Blot (also called protein immunoblot) is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract.

It works by leveraging the power of antibody-antigen specificity, allowing researchers to identify a single protein within a complex mixture of proteins.

The Core Principle: Separate proteins by size → Transfer them to a stable membrane → Use antibodies to "probe" for the protein of interest.

1. Western Blot By: Moaz Maher Supervised by : Dr.Walid Lotfy

2. defination

3. Definition Western blotting is a laboratory technique to detect specific proteins from a cell or tissue sample The name Western blot was given to the technique by W. Neal Brunette there are variations of the WB and one can optimize the protocol for specific application, in the majority of cases WB is still widely used in its basic form with only subtle adjustments mainly in the generation and characterization of the antibodies used

4. Application Western blotting (WB) is widely used in diagnosing and studying several diseases where protein expression or immune response is a critical factor Western blot analysis useful in detecting antibody or protiens which confirm disease diagnosis Like : HIV/AIDS Lyme disease Prion Diseases Hepatitis B Usually used as confirmative test for Hiv infection after ELISA test which is also used to detect proteins but not specific as WB

5. Steps of WB TECH Separation Detecting Transfer

6. Steps of WB TECH

7. Steps of WB TECH Separation : Proteins are separated by size using gel electrophoresis, where an electric field moves proteins through a polyacrylamide gel matrix based on their molec Transfer :The separated proteins are transferred from the gel onto a membrane (usually nitrocellulose or PVDF), which captures the proteins in their separated positions for detection . Detection :Specific antibodies bind to the target protein on the membrane, and a secondary antibody linked to a detectable enzyme or tag reveals the protein’s location, typically via chemiluminescence or colorimetric signals.

8. 1 st step separation after preparation

9. 1-Sample Preparation: 1-Sample preparation require to lyse cells or tissues using mechanical or enzymatic homogenization to extract and solubilize protein using detergents 2-it s mandatory to use protease inhibitor to prevent protein degradation and kept at 4 Celsius degree. 3- SDS ionic surfactant is added to give protein net negative charge, unfolding protein make it linear and ready to separation according to size 4- B oiled in sample buffer at 95 dgree Celsius for (5-10) min for denaturation

10. 2. Gel Electrophoresis The sample is loaded onto a polyacrylamide gel ( PAGE ) after adding molecular weight size marker then subjected to an electric field, lead to separating proteins based on molecular weight, with smaller proteins migrating faster through pores of gel.

11. Types of gel There are 2 main types of gel Stacking gel : lower percentage of acrylaldamide lower PH different ionic composition Which help to compress protein Gradient (resolving) gel : Concentration of acrylaldamide progressively increase

12. 2 nd step Transfer (Blotting)

13. 3.Electrotransfer (Protein Transfer) Separated proteins are transferred from the gel onto a (solid support ) membrane (nitrocellulose or PVDF), preserving their separated pattern and making them accessible for antibody binding.

14. Wet Transfer: This method immerses the gel and membrane in a buffer solution and applies an electric field to transfer proteins from the gel to the membrane. Wet transfer generally provides high efficiency for large proteins but takes longer and requires more buffer. Semi-Dry Transfer : In this method, a minimal amount of buffer is used between the gel and membrane layers within a stack, making the setup compact and faster. Semi-dry transfer is efficient for smaller proteins but may not work as well for large proteins due to limited buffer volume​

15. 4. Blocking The membrane is blocked with a protein solution to prevent non-specific binding of antibodies, reducing background noise in the results.

16. 3 rd step Detection

17. 5-Probing Probing: Probing in Western blotting involves using specific antibodies to detect target proteins on the membrane Can be done in one step or 2 steps

18. Primary Antibody Probing : The primary antibody (non-labeled) binds to the target protein on the membrane, acting as the main detector by directly recognizing unique epitopes protein parts

19. Secondary Antibody Probing: The secondary antibody binds to the primary antibody and is typically labeled with an enzyme (like HRP) or a fluorescent tag . It amplifies the detection signal

20. 6-Washing Washing: Washing removes unbound antibodies from the membrane after each incubation step, reducing background noise. It enhances the specificity and clarity of the detected signal in Western blotting

21. 7-Detection Detection: Detection in Western blotting involves direct visualizing the target protein using fluorescence . Another method include chemiluminescence allows better quantification.

22. 8-stripping (optional ) Stripping: Stripping removes bound antibodies from a Western blot membrane, allowing reprobing with different antibodies on the same sample. This enables analysis of multiple proteins on a single membrane, conserving samples and reagents.

23. Assignment

24. Aim of the study The study aimed to determine if DBS samples could be used with commercial ELISA and Western blot tests to detect HSV-2 antibodies . Benefit : DBS samples are beneficial because they are easier to collect , store , and transport than serum sample

25. Method Methods : A total of 48,067 people were selected with an age range of 15–49 years Then study used a sub-sample of 908 individuals with an over-representation of samples with index(S/CO) from 2.0 to 6.0, from sexually active subjects

26. First results without modification Elisa results on DBS were very similar for Serum sample Serum Samples : The S/CO index values ranged from 0.289 to 7.248 for the 13 serum samples. Six samples had values between 0.0 and 2.9 (considered negative to low-positive ), while seven samples ranged from 3.0 to 8.0, indicating higher antibody levels ( positive ) DBS Samples : For the corresponding 13 DBS samples (eluted in 400 µl buffer), S/CO values ranged from 0.487 to 7.866 . Five samples had values between 0.0 and 2.9 , and eight samples were between 3.0 and 8.0 , showing a similar distribution to the serum samples . BUT after confirmation with WB : all results appear negative So the need to make some modifications

27. Modifications ELISA and western blot Test Modifications : adjusting the incubation times and dilution process to increase antibody detection. Cutoff points modified using ROC analysis to improve the test's sensitivity and specificity for Elisa

28. Results after Modifications In the Western blot analysis: Serum Results : Out of 14 serum samples, eight tested positive for HSV-2 antibodies, and six were negative . DBS Results : Initially , all DBS samples at a 1:51 dilution were negative . And after changing to 1:5 dilution produced results very similar with the serum samples According to the study results with the new cut off points for ELISA, the sensitivity and specificity values were near of 95 %

29. conclusion DBS samples , when tested with modified ELISA and Western blot procedures, can be used to detect HSV-2 antibodies effectively. Thus can be alternative for serum sample in detection Hsv-2 antibodies

30. Criticism The study applied on a small number of serum and DBS samples that may not giving all variation between serum & DBS DBS samples are susceptible to hemolysis during the transporting and no data about collecting time The study mainly relied on adjusting cutoff points may lead to bias ELISA results always needed to be confirmative with WB - specially the undetermined results - which is high cost. No funding for the study may limit the resources .

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