Western Blot Detection of HIV-1 gp120 10/16 Honors BioMed 2 Redwood High School

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1 Western Blot Detection of HIV-1 gp120 10/16 Honors BioMed 2 Redwood High School Background THE BIOLOGY OF AIDS/HIV Acquired immune deficiency syndrome (AIDS) is a disease characterized by the progressive deterioration of an individual s immune system. The immunological impairment allows infectious agents such as viruses, bacteria, fungi and parasites to invade the body and propagate unchecked. In addition, the incidence of certain cancers dramatically increases in these patients because of faulty immunesurveillance. AIDS is a serious threat to human health and is a global problem. Intensive research is being done to advance methods of detection, clinical treatment and prevention. THE HIV VIRUS Duplication of this document, in conjunction with use of accompanying re tory use only. This document, or any part, may not be reproduced or dis written consent of EDVOTEK, Inc. Copyright 2005, 2011, 2012 EDVOT 2012_02_24AM The Biotechnology Education Company EDVOTEK ww The AIDS etiologic agent is the human immunodeficiency virus type 1 (HIV- 1), a retrovirus. HIV-1 contains an RNA genome and the RNA-dependent- DNA-polymerase termed reverse transcriptase. Members of the retrovirus family are involved in the pathogenesis of certain types of leukemia and other sarcomas in humans and animals. The structure and replication mechanism of HIV is very similar to other retroviruses. HIV is unique in some of its properties since it specifically targets the immune system, is very immune-evasive, forms significant amounts of progeny virus in vivo during initial stages of infection and can be transmitted during sexual activity. The HIV viral particle is surrounded by a lipid bilayer derived from the host cell membrane during budding. The viral proteins are identified by the prefix gp (glycoprotein) or p (protein) followed by a number indicating the approximate molecular weight in kilodaltons. The lipid bilayer contains gp120 and gp41. These two proteins are proteolytic products of the precursor gp160. The gp41 anchors gp120 in the bilayer. The protein gp120 is routinely used as a diagnostic marker for HIV in Western Blot Analysis. More recently other viral gp proteins are also included in the test. Beneath the bilayer is a capsid consisting of p17 and p18. Within this shell is the viral core. The walls of the core consist of p24 and p25. Within the core are two identical RNA molecules, 9800 nucleotides in length. Hydrogen bonded to each viral RNA is a cellular trna molecule. Viral RNA is coated by tightly bound molecules of p7 and p9. The core also contains approximately 50 molecules of reverse transcriptase. There are several other viral proteins whose precise functions are not fully understood. The virus can be grown in tissue culture for diagnostic and research purposes. Several of the viral proteins have been cloned and generated in relatively large quantities. An individual can receive an inoculum of HIV through an abrasion in a mucosal surface (e.g., genital and rectal walls), a blood transfusion, or by intravenous injection with a contaminated needle. Virus or virally infected cells are found in body fluids such as semen and blood. The most important target for the virus is hematopoietic cells such as bone marrow derived monocytes, myelocytes and lymphocytes. Infection of immune system effector cells such as T cells and macrophages ultimately produce the most profound clinical consequences. Gp120 binds to the CD4 receptors on the surface of T helper (T H ) cells. These receptors are membrane bound glycoproteins involved in T cell activation. Under normal conditions CD4 acts as a receptor for major histocompatibility class II (MHC II) membrane bound molecules that are present on the surface of macrophages and several other types of cells. TH cells are required for the body s overall immunological responses. The viral lipid bilayer fuses with that of the cells membranes and the viral protein capsid becomes internalized via receptor-mediated endocytosis. Subsequently, the rest of the CD4 receptors are down-regulated and gp120 appears on the T cell surface. Through a complex mechanism, the reverse transcriptase synthesizes a double stranded DNA copy of the genomic RNA template. The trna molecule acts as the primer of the first strand synthesis. The RNAse activity of the reverse transcriptase degrades the RNA strand of the RNA-DNA duplex and the enzyme synthesizes a complimentary DNA strand. The DNA reverse transcripts (double-stranded DNA) migrate Background In Glycoprotein Lipid bilayer Reverse transcriptase THE HIV VIRUS The AIDS etiologic agent is the human im 1), a retrovirus. HIV-1 contains an RNA g DNA-polymerase termed reverse transcri family are involved in the pathogenesis other sarcomas in humans and animals. anism of HIV is very similar to other retr properties since it specifically targets the sive, forms significant amounts of proge of infection and can be transmitted duri The HIV viral particle is surrounded by a cell membrane during budding. The vira fix gp (glycoprotein) or p (protein) follow proximate molecular weight in kilodalto and gp41. These two proteins are prote gp160. The gp41 anchors gp120 in the b Protease Protein coat RNA Core Integrase ly used as a dia Blot Analysis. M are also include is a capsid cons shell is the viral of p24 and p25 RNA molecules drogen bonded molecule. The molecules of p7 proximately 50 There are sever functions are n grown in tissue purposes. Seve cloned and gen

2 into the cell nucleus where they become covalently integrated into the cellular genomic DNA. The integration is partly catalyzed by viral proteins. The copy DNA integrates via specific, self-complimentary sequences at both ends called long terminal repeats (LTRs). These sequences also have important functions in viral transcription. The integrated copy DNA is called proviral DNA or the provirus. The provirus enters a period of latency that can last for several years. The proviral DNA is replicated along with the cellular DNA and can be inherited through many cell generations. The HIV proviral DNA contains the major genes common to all non-transducing retroviruses. These genes are gag, pol and env. HIV-1 also contains five or six smaller genes. Unlike other cellular DNA polymerases, HIV DNA polymerase (reverse transcriptase) has a high error rate. These frequent mutations continually change the viral protein epitopes. This is believed to be the main mechanism of HIV immunoevasion. The gag gene is translated into a polypeptide that is cleaved by a viral protease into four proteins that form the inner shells. The protease is encoded in the pol gene. The pol gene encodes the reverse transcriptase and the integrase that is responsible for the genomic incorporation of copy DNA. The env gene encodes the surface glycoproteins the viral particles acquire as they bud from the cells. Viral replication causes the destruction of the TH cells. Enzyme linked immunoadsorbent assay (ELISA) is an important immunochemical method used for the detection of low levels of antigens. ELISA is used for clinical screening for HIV in the blood supply. ELISA testing for HIV detects the patient s circulating IgG directed toward the viral antigens. A positive reaction in the ELISA requires more definitive testing for verification by the use of a Western Blot. One reason for this problem is that anti- bodies sometimes exhibit cross reactivity. PROPERTIES OF PROTEINS Denaturing SDS gel electrophoresis separates proteins based on their size. SDS (sodium dodecylsulfate ) CH₃(CH₂)₁₁OSO₃Na is a detergent that consists of a hydrocarbon chain bonded to a highly negatively charged sulfate group. SDS binds strongly to most proteins and causes them to unfold to a rod like chain and makes them net negative in charge. In the absence of a denaturing agent such as 2-mercaptoethanol, no covalent bonds are broken in this process. Therefore, the amino acid composition and sequence remains the same. Since its specific three-dimensional shape is abolished, the protein no longer possesses biological activity. Proteins that have lost their specific folding patterns and biological activity but have intact polypeptide chains are called denatured. Proteins that contain several polypeptide chains, which are non-covalent bonds will be dissociated by SDS into separate, denatured polypeptide chains. Proteins can contain covalent cross-links known as disulfide bonds. These bonds are formed between two cysteine amino acid residues that can be located in the same or different polypeptide chains. Treatment of proteins at 100 C for 3 minutes in the presence of high concentrations of reducing agents, such as 2-mercaptoethanol, will break disulfide bonds. This allows the SDS to completely dissociate and denature the protein. During electrophoresis, the SDS denatured proteins migrate through the gel towards the positive electrode at a rate that is inversely proportional to their molecular weight. In other words, the smaller the protein, the faster it migrates. The molecular weight of the unknown is obtained by the comparison of its position after electrophoresis to the positions of standard SDS denatured proteins electrophoresed in parallel. THE WESTERN BLOT Western Blot Analysis involves the direct transfer of protein bands from an agarose or polyacrylamide gel to a specially formulated nitrocellulose membrane for analysis. Following electrophoresis, the gel is removed from the tray and the membrane is placed directly on the gel. Protein bands are transferred to the surface of the membrane and are adsorbed on the membrane by hydrophobic bonds. This transfer can be achieved electrophoretically in specially designed chambers, by capillary flow, or by the application of vacuum. 2

3 The total protein transferred can then be visualized by staining the membrane with protein dyes. Visualizing a specific protein within a mixture of proteins can also be detected by immunochemical methods. For immunological detection of specific protein, the unstained membrane is placed in a blocking buffer that contains detergent and protein that bind to all unoccupied sites on the nylon membrane. The membrane is then incubated in buffer that contains antibody to one (or more) of the blotted proteins. The antibody binds to the adsorbed protein (antigen) and subsequent washings removes unbound antibody. A secondary antibody that is covalently linked to an enzyme such as alkaline phosphatase or horseradish peroxidase is used for detection. The conditions for cross-linking the enzyme to the secondary antibody does not appreciably affect the antigen binding specificity, the affinity of the antibody, or the catalytic activity of the enzyme. The membrane is then incubated in a solution of the secondary antibody where it will bind selectively to the bound antigenprimary antibody complex. Following this treatment, the membrane is washed to remove the unbound secondary antibodyenzyme complex and is then incubated in a solution containing a phosphatase or peroxidase substrate. The products of the enzymatic reaction yield chromogenic products that are easily visible on the nylon membrane. In this lab we will use a modified Western Blot protocol to detect gp120. Lymphocyte tissue cultures were exposed to serum from three HIV positive individuals as judged by ELISA testing. The cells were grown in tissue culture. The cell growth media, which should contain virus, is collected and concentrated. Samples are prepared for electrophoresis by boiling the concentrate in buffer containing SDS and 2-mercaptoethanol. After transfer to a nitrocellulose membrane Western Blot Stain is used to detect any gp120 present. The size of the viral protein can be confirmed by comparison to Standard Molecular Weight Dye Markers. Protocol Day 1 * We are only using the middle six wells of the gel. As a result, you will be leaving the 1st and 8th wells empty. For our purposes, we will call the 2nd well in the gel well no. 1, making the 7th well in the gel well no. 6. Load Samples into Wells 1. Load 20 µl of Sample A, the positive control, into well no Load 20 µl of Sample B, the negative control, into well no Load 20 µl of Sample C, Patient 1, into well no Load 20 µl of Sample D, Patient 2, into well no Load 20 µl of Sample E, Patient 3, into well no Load 20 µl of Sample F, Standard Molecular Weight Dye Markers, into well no. 6. Running the Gel 7. After the samples are loaded carefully slide the gel box cover onto the electrodes. 8. Insert the plug from the black wire into the black input of the power supply (negative input). Insert the plug from the red wire into the red input of the power supply (positive input). 9. Set the power supply to 75 volts and run the electrophoresis for 80 minutes. The blue running dye must travel at least 4.5cm from the wells (leading edge of the wide red line) 10. When the gel has approximately 15 minutes left to run, set up your Western Blot. Setting Up The Western Blot 3

4 11. Place a piece of plastic wrap on the bench top you have designated as your blot station. Smooth the plastic wrap as the filter paper, gel, membrane, and cotton pads will be placed onto it to make the blotting sandwich. 12. Put on a pair of powder-free latex gloves. Areas of the membrane touched by ungloved hands will absorb oil residues creating areas on the membrane that will not bind to the proteins during transfer. 13. Use a forceps to carefully transfer a Western Blot membrane to a staining tray labeled with your name or initials. Do this by holding a Western Blot/blue cover sheet complex in the palm of your hand. Use a forceps to remove the top protection sheet; Continue to use the forceps to lift the white Western Blot membrane off of the bottom cover sheet. Transfer the membrane to the staining tray. 14. Pre-wet the membrane by poring enough methanol into the tray to cover the membrane for 10 seconds. Pour off the methanol into the methanol beaker, to be saved. 15. Immediately pour enough distilled water into the tray to cover the membrane for 5 minutes to remove the methanol. Pour off the water into a waste beaker. 16. Pour enough transfer buffer into the tray to cover the membrane. Allow the membrane to remain immersed in the transfer buffer until the membrane is needed in step # Set up two additional staining trays. Building The Western Blot 18. When the gel is done running, turn off the power supply, unplug both power cords and remove the top of the gel box. Reach into the gel box and lift out the tray containing the gel. Carefully transfer/slide the gel into one of your extra staining trays. 19. Immerse the gel in transfer buffer for 15 minutes. Perform procedure No. 20 when the gel has two minutes left to soak. 20. Place one piece of filter paper into your third staining tray. Saturate the filter paper with transfer buffer. Remove the filter paper and place it on the plastic wrap at your blot station. 21. When the gel is done soaking decant as much transfer buffer as possible (into your waste beaker) without damaging the gel. Carefully transfer the gel from the staining tray and place it UPSIDE DOWN evenly on the saturated filter paper. Although the gel may extend off the filter paper, make sure that the six center lanes are on the filter paper. 22. Carefully roll a 1ml serological pipet over the surface of the gel to remove any air bubbles trapped under the gel. 23. Pipet 2 ml of transfer buffer over the top of the gel (make sure that the six lanes in the middle of the gel get covered first. Transfer the Western Blot membrane onto the gel. Make sure that the membrane is symmetric on the gel - covering the middle six lanes and the wells (align the top edge of the membrane with the top edge of the wells). Roll a pipet over the surface of the membrane to remove air bubbles. 24. Use a pencil to lightly trace the outline of each of the bands in lane number. 6. Beside each mark, indicate the color of the respective band. (B1 = Blue 1, B2 = Blue 2, P = Purple, and R = Red). 25. Saturate another piece of filter paper with transfer buffer and cover the membrane with the wet filter paper. Roll a pipet over the surface of the paper to remove air bubbles. 26. Add a second piece of dry filter paper to the top of the stack. Remove air bubbles. 27. Evenly place 6 cm stack of cotton pads on top of the stack. 28. Rinse and dry the tray that contained the gel as it soaked. Set this tray on top of the stack and place a light-weight beaker (400ml size) in the tray. Cover the entire stack/set-up with plastic wrap. Allow the protein transfer to proceed overnight. Day 2 4

5 Processing and Staining of the Blot Membrane 29. Remove the tray, cotton pads and filter paper from the top of the membrane. Leave the membrane in place on top of the gel. 30. Using a pencil, lightly trace the outline of each well and number them according to loading sequence. Remember that the gel is upside down. 31. Using forceps, remove the membrane from the gel and place it on a cotton pad. The side that was in contact with the gel should be facing up. 32. With a pencil, on one of the lower corners write "F" (for front). On the other corner, write your initials. Place the membrane on the cotton pad in a 65 C incubation oven for 10 minutes to fix the samples. 33. While the membrane is cooking secure three staining trays, latex gloves, and one Protein InstaStain card. Place enough methanol in one tray to immerse the membrane. Place enough distilled water, in the second tray, to immerse the membrane. Place enough stain solvent into the third tray to completely immerse the membrane. 34. Once out of the oven, immerse the membrane in the methanol just long enough so as to assure that there are no dry areas. 35. Transfer the membrane from the methanol to the distilled water tray for 5 minutes. 36. Transfer the membrane from the distilled water to the stain solvent. The membrane should be completely covered. Wearing gloves, gently float your Protein InstaStain card above the membrane with solid purple side down. Let the membrane soak for 15 minutes, with slight agitation each minute. NOTE: if the color of the membrane remains light, allow the membrane to soak longer until it is dark blue. 37. Using forceps, remove and discard the stain card, and transfer the membrane back into the methanol tray. Gently rock the membrane in the alcohol to de-stain. Look for the positive control to appear. When the bands are evident, immediately transfer the membrane into the fresh distilled water tray, and cover with plastic wrap. LABEL YOUR TRAY. Day 3 Data Collection and Protein Size Calculation 38. Using a forceps remove the membrane from the distilled water and lay the membrane on a cotton pad. Do not touch the face of the membrane with your fingers smudging will occur. Compare the three patient samples to the positive and negative controls. Create a data table and sketch and label a pictorial of your membrane. 5

6 Molecular Weight Estimation of HIV gp120 Protein [one graph per group] 1. Measure the distance (mm) of each band traced on the membrane for the standard markers and the positive viral samples. Each measurement should be from the bottom of the well to the bottom of each band (defined as the bottom of the darkest, most solid portion of the band regions that are flaring do not count) The following are the known molecular weights for the standard dye markers: B-1: 140,000 daltons (140Kd) B-2: 110,000 daltons (110Kd) P: 90,000 daltons (90Kd) R: 70,000 daltons (70Kd) 2. Plot the standard marker data using semi-log graph paper (provided). The distance traveled (mm) should be plotted on the x-axis, while the molecular weights should be plotted on the y-axis. Label each point and each axis. Based on the plotted points for the molecular weight markers, produce a line of best fit. Additionally, create a small data table on the graph paper displaying distance traveled and estimated molecular weight. 3. Determine the molecular weight of the viral protein by extrapolating from the standard curve produced from the standard marker data. Note this on your graph. 4. Staple your group membrane to your individual graphs and turn in as a group packet. Lab Note: You will revisit the background information on the final exam. Be sure that you understand the information from each section of the background. 6