The aim of this procedure is to study proteomic expression variation in renal cell cancer using reverse phase protein arrays or RPPA. This is accomplished by first mapping and selecting heterogeneous areas of individual tumors. The second step is to extract proteins from the samples.
Next, the proteins are normalized and printed on RPPA slides. The final step is to analyze the slides using antibodies against proteins of interest. Ultimately, RPPA is used to show differential proteomic expression variation in two treatment groups of renal cell cancer.
This method can help answer some of the key questions in the field of renal cell cancer. For great protein heterogeneity exists. The implications for this technique are that it may impact positively on the treatment of patients with renal cell cancer who are treated with targeted therapies.
This is because we can investigate both pre and post-treatment heterogeneity in tissue samples, and therefore hopefully identify novel druggable targets for patients who develop acquired resistance to these treatments. Demonstrating the cryostat and H knee procedure will be research lab manager Helen Caldwell, demonstrating the protein extraction technique will be research technician from our group, Jo Nanda. Demonstrating the OR PPA printing will be our colleague from St.Andrews, Peter Mullen.
To begin this procedure, remove tumors from the minus 80 degrees Celsius freezer and keep on dry ice. Divide tumors into sections of approximately one centimeter cubed. Map the original position of each tumor section relative to each other and label with a unique name.
Coat samples in OCT and cut in a cryostat of minus 22 degrees Celsius after the samples are stained. Using the hemat, toin and DSN counters staining method, analyze the frozen sections by microscopy. Ensure the sections are a viable tumor of clear cell renal cell carcinoma and grade them as high grade, low grade or mixed low and high grades.
Select up to four samples from each morphologically differing region within each tumor for protein extraction, which will be demonstrated next to extract protein from tumor samples. Place 50 to 75 milligrams of tissues. Cut from a CT into two milliliter tubes.
Add 990 microliters of lysis buffer, supplemented with a protein in and phosphatase and protease inhibitors. Add a single five millimeter steel ball to each tube and homogenize at 50 hertz for five minutes. Using a tissue laser.
Check the level of homogenization and then homogenize for another five minutes. Using a pipette transfer homogenized sample to a new two milliliter tube, leaving the steel ball behind a 10 microliters of Triton X 100 to each sample, and then centrifuge at 13, 000 times G for 30 minutes at four degrees Celsius. Transfer the S supernatants to fresh micro centrifuge tubes and determine protein concentration using the BCA assay.
Normalized protein concentrations to one milligram per milliliter prior to our PPA printing. A series of five twofold dilutions are made from each sample. One of the most time consuming aspects of this procedure is making hundreds of dilution successful outcome can be assured by double checking prior to printing slides.
A microgrid two robotic spotter is then used to spot protein lysates onto nitrocellulose coated glass slides. Each sample is spotted in triplicate, resulting in a total of 15 spots per sample. The slides used in this experiment contained two pads onto which the samples are spotted.
Each pad was spotted with identical samples, in this case, 100 samples. Other available formats include one eight and 16 pad slides. The higher the number of pads, the smaller the number of samples that can be onto each.
To begin the procedure for protein detection wet slides, an excess blocking buffer and incubate room temperature For one hour on a rocking platform, prepare 800 microliters of primary antibodies in blocking buffer at the desired concentrations and keep on ice. After the one hour incubation. In blocking buffer mount slides in either the single frame chip clip or the fast frame four base slide holder so that a tight seal is formed between the slide and the incubation chamber.
Remove residual buffer from wells and add 600 microliters of primary antibody to the respective wells. Place the slides and chamber into a sealed wet box and incubate on a rocking platform overnight at four degrees Celsius on the following morning. Remove slides from the cold room.
Carefully remove the primary antibodies from each. Well add 600 micro releases of 0.1%PBS between 20 or PBST and wash slides on a rocking platform at room temperature for five minutes. Replace the PBST with fresh PBST in this way.
Wash slides a total of three times. Remove buffer from wells and add 600 microliters of fluorescently labeled secondary antibodies at the appropriate dilution to the respective wells. Incubate with secondary antibodies at room temperature for 45 minutes with gentle shaking, it is important to protect the membrane from light until the time it is scanned.
After 45 minutes, remove secondary antibodies from wells and briefly wash three times in 600 microliters PBST at room temperature. Remove slides from the carrier, transfer to a suitable container and wash in excess PBST for 15 minutes. Keeping the membrane in the dark.
Remove PBST and wash the membrane with PBS at room temperature for 15 minutes to remove residual tween 20 again, keeping the membrane in the dark. Try the fast slides in a 50 degree Celsius oven for 10 minutes. Finally, scan the slides as 680 nanometers and or 800 nanometers depending on the secondary antibodies used.
Make sure to keep the slides in the dark until they have been scanned. Save images as T files. An example of a scanned RPPA slide can be seen in this figure with both 680 and 800 nanometer channels shown.
Separating the images by wavelength enables each pad on the RPPA slide to be analyzed and individual protein expression determined as shown here. The expression of individual proteins across the samples is unique with gel soin having a high level of expression across the slide compared to cmic, which has a much lower protein expression, but still universally expressed across all the samples tested. In contrast, CD 10 gave variable expression with certain samples giving a high level of expression despite other samples having little or no expression detectable.
Finally, p JK two failed to produce any detectable protein expression above background level. This next figure highlights the lack of cross reactivity between secondary antibodies and primary antibodies of another species. The image on the bottom left shows detection of protein expression using secondary antibodies scanned at the correct wavelength of 680 nanometers.
Protein expression is not detected in the image on the right, scanned at the incorrect wavelength of 800 nanometers. An example of the super curve used for comparative analysis of all the samples on the RPPA slide is shown here. The green spots represent acceptable spots and red spots represent outliers based on predefined limits In the analysis software.
The super curve is used to produce relative protein-based expression data. This figure shows the expression levels of a representative protein mlh one across three RPPA slides highlighting how RPPA can detect differences between treated and untreated samples. Each sample here corresponds to a tumor subregion.
This next figure shows differential protein expression for mlh one after the samples have been grouped based on treatment. Higher MLH one expression was found in the pre-treatment group when the intratumoral variance in mlh one expression between pre and post-treatment samples is calculated. After grouping the samples based on treatment, a higher variance was observed in post-treatment samples.
After watching this video, you should have a good understanding of how to utilize or PPA for the study of protein heterogeneity.
