 As we started discussing about reverse phase protein arrays and Dr. Ramesh Umani a guest scientist provided you an overview of how to use this technology for various applications. So today Dr. Umani will continue his lecture and he will talk to us more about this novel technology reverse phase protein arrays. In today's class you will learn about how to choose the correct samples for the experiment, what are the methods for sample preparation, selection of antibodies, spotting methods and signal detection methods. So let us have Dr. Ramesh Umani to continue his lecture now. First is samples there is no limitation any sample can be analyzed by RPMS or any sample which can be analyzed by western blot can be analyzed in RPME. Only thing is you have to follow certain methods to prepare your clean sample to make it ready for RPME approach. It is a high throughput technology that performs protein assays on thousands of samples simultaneously including tissue lysates just microdesected tumor samples there at least you will have a chance to get some microgram level proteins, some couple of milligram level proteins and then laser capture microdeception LCM. There you end up with very very low quantity of sample you cannot perform n number of western blots to validate n number of targets and PFE fixed tissue lysates also you can do. So there are separate methods are available to make the protein extracted from formalin fixed tissues to use for RPMS. Sometimes those samples cannot be analyzed by western blotting approach you see smearing effect which can be avoided here. The cell lysate particularly our tumor cell lysate every one of you might be handling some of the cell lines in the lab so the lysates can be prepared and small number of isolated stem cells are the other rare cell types. So now stem cell biology is a very very attracting word but they will deal with always thousands of cells not even millions of cells. We do millions of cells in cancer research serum also can be spotted there plasma or any body fluids such as cerebrospinal fluid or urine can be directly printed on the lysate by following specific methods. So to print the samples how to prepare the samples is very simple cartoon here can take a lysates buffer put the tissue of the rest and homogenate and spin down the tube and take the supernatant and go for printing or can kept in the minus 80 go home and come back tomorrow. This is a way but it is not that as simple as just preparing there are multiple steps has to be taken care. So you have to plan experiment and take all the parameters into consideration before executing the experiment. So in that way this is also one of the critical point. So this critical point basically highlights what kind of buffer can be chose not all proteins in all samples can be solubilized by single buffer. This is a major limitation in array approach. So lysates buffer should be optimal to work for maximizing the RPPS signal. So it should not interfere with the visualization of signals using any labeling system you follow either secondary antibodies like IR labeling, HRP label or alkaline passphrase label or directly Psi3 or Psi5 label and it should be compatible with the detection methods like a calorimetric amino luminescence and fluorescence methods should not interfere with substrate on solids and absorption of lysates. See for example you might have performed washing steps. What if the buffer is not compatible and washed away along with the proteins spotted on the glass slide end of the day you will have a red face no signals. So definitely should not interfere with the substrate on the slides in this case nitrocellulose substrate and should not interfere with adsorption of the lysate. I mean that means attachment of the or immobilization of your protein sample. Extraction efficiency may vary with the different lysate reagent and is dependent on the tissue being examined. Let us say for example if you are taking a prostrate tissue which will have a prostrate some stones will be there then you need a stringent buffer. Let us say for example you are lysing the cell lines simple rippa buffer is sufficient or ampere buffer is sufficient. So lysate reagent should be supplemented with freshly added protease inhibitors. Of course if you wanted to understand the post translational modifications and you need to have the inhibitor cocktails like phosphatase inhibitors and also some of the protease inhibitors not to degrade the protein of interest. So with my experience at least I worked with these four different buffers definitely these four different buffers behaved entirely different with the different samples believe that any one buffer will help is not true. In my hands we had a different experience with different buffers and different tissues and different cell lines. So again one of the volunteer sitting back was emphasizing that this method is always to be optimized in your own lab in your own hands in your own setup. So this there is no hard and fast rule that so and so teacher explain this concentration it will work it will not work. So you have to optimize please keep this in mind. Then second point highlighted was the essential requirement is the good quality antibodies. So here you need to spend at least some couple of months to years to establish antibody panel in your lab. At least we could establish I mean with my postdoc supervisor we could establish 190 antibody panel in one year from 3000 antibodies purchased from the market. You can see that 2800 plus antibodies just went into dustbin of the German Cancer Center that time. So now those antibodies we are repeatedly purchasing and we are using it but this effort one year effort helped in the long run definitely how it is I will highlight you here. Our approach for antibody validation is like this starting point take that western blot through western blotting approach take 10 different cell lysates or from more cell lines run 10 different lines then do western blot. If the antibody gives a single band say yes go to next step and then you pull the lysates from 10 or more different cell lines run a single line that means in a complex mixes also antibody has a capacity to recognize a protein of interest without any non-specificity. If it gives a single band it will go to the additional validation steps by western blot using knockdown experiment and stimulation experiments and if it is and if it is yes then it will go to the validation by RPPA. In RPPA we will validate by serial dilution of the samples whether antibody is able to detect the serial dilution the 2 fold 10 fold dilution properly or 5 fold dilution properly. If it is yes then this correlated is if the RPPA data is correlated with expect results from protein of interest then we declare that it is useful for RPPA and also every antibody will get a 3 codes go ahead use with caution and not suitable. These are the 3 different words in RPPA community we keep on hearing so antibody they write code so it is like a go no go then use caution these are the 3 words we exchange by emails. You can see that this is a like a pain we used to run these kind of western blots several hundreds to thousands for validation purpose you can see that some of the bands are very nicely giving single bands some of them are like a non-specific bands and here you can see that this is completely crap you cannot use them and mostly with my experience I am not as again brand ambassador for any antibody companies here at least I had a good experience with cell signaling very bad experience with Santa Cruz. So, I do not get offended please I just it was my experience I am sharing with a very lighter note with the student community here. So, you can see that if you have a single band here directly it is yes then we will proceed to this pipeline here. So, once we reach this step we need to follow additional steps this kind of antibodies we will grab them first step itself we do not spend any time and we will directly buy the another antibody. So, next round of validation is that again using western blot we take a suitable cell line and stimulate them with some kind of factors where your protein of interest gets modulated you know it prior information already and you look for this known information in the western blot results. In this experiment what we have done is we have taken HEPG to cell line and we stimulated with EGF and insulin these two factors will stimulate the phosphorylation of AKT and phosphorylation of ARC protein and those phosphorylations have been observed or not these are also mutually exclusive you can see that EGF is stimulating here phosphorylation insulin is not doing. So, this kind of approach we follow this is one example I am showing you. So, with this approach it is passed then it will go into the next line. Then we will not leave that story there itself again we take those antibodies and this pass for specific antibodies choose the different cell line whether they are behaving in the same way or not. So, here you can see that with the insulin pass for drug to pass for relation is going up with EGF only EGFR is going up. So, again these are the mutually inclusive exclusive garbage in garbage out approach basically to tell you in a very crude manner. So, once we have this we will not leave the story there again we have now SIRNA available for all the proteins expressed in the orpheum we buy the SIRNA down regulate them transfer to the cells make the lysates random on a western blot whether this antibody is really detecting the down regulation or not. So, in this case we have given to the cells SIRNA against EGFR and our antibody is detecting the down regulation of EGFR. So, it has passed through the this step here knock down or perturbation experiments. So, after this then we validate them by reverse phase protein or we simply take very pool of lice small pool of lices print on the glass slides and use this antibody and probe it see that whether it is really detecting the signals or not. So, when you plot the results like concentration versus signal intensity. So, as increasing the concentration. So, the signal is going up blank signal is at the base level the signal intensity is fine here and also to see that the detection levels are really in the range of detectable or not. So, target signal versus blank signal if the target signal is very near to blank signal there is no point we do not get a linearity there. So, then we have to drop the antibody at this step after reaching here if we see no here then we have to go here again. So, this is a problem after this process if we for process these two filters we have a like mono specific antibodies in this example I am showing Paso P38. This antibody is go for RPPA approach and antibody is approved for reverse phase protein or a approach you also need to do one more step you need to see the correlation between western lot and RPPA results. Because many of the why I am emphasizing this was that one of the RPPA conference many people did not believe that RPPA is better than the western lot. So, that was the reason. So, this paper was published in clinical cancer research where the RPPA data is very well correlated with the western lot data. So, the same sample set the RPPA data is generated compared with the western lots generated from the same sample set. So, then it is clearly understood that the community accepted that the RPPA data will correlate with the western lot data. So, that it is useful to validate large number of proteins in a smaller quantity of samples. So, here we have spotted the I mean this group Gordon Mills from MD Anderson they spotted serially diluted tumor lysates and it western lots and normalize the data this outcome here. So, now we have the antibodies suitable for reverse phase protein array approach. So, at this point to educate you in a different way not all antibodies agnest one target will behave the same way. Let us say for example, if you search cell signaling website for a EGFR or PASFO EGFR antibody target will have a 10 different antibodies with the different catalog numbers. That means, they are they are from different clones or different poly different monoclonal antibodies and not all the antibodies behave in the same way how it is now you look at this picture. So, in one lysate we used for downregulation of EGFR from the same lysate we did a western lot in a serial dilation manner and used two different antibodies to detect the signals here. Two different antibodies nicely showed the down concentration dependent signals for EGFR and also western lot results shows that 80 percent downregulation happened for EGFR after adding SARN. This data is completely wrong how it is when you did RPPA with the same samples can see that this is the anti EGFR one antibody detecting the lysate where EGFR is downregulated and here are all control samples. In this case this is antibody 1, antibody 2. When you look at the correlation this first antibody detects 48 percent downregulation second antibody says 83 percent downregulation. Now again question mark. So, when we deal with this antibody related approach or antibody based approach we should be very very cautious in choosing the antibody. Otherwise end of the experiment we not able to conclude or not able to infer any meaningful data. So, this is what again all antibodies cannot detect the single protein in the same way like for example, in this slide you can see the stat 3 antibody different stat 3 antibodies detecting the stat 3 in the same sample in different manner. You can see LOD this limit of detection some numbers are written here definitely I think I will circulate the slides also to you if you cannot note the numbers you can go through later on. So, this is green is a polyclonal red is a monoclonal blue is a blank values you can see that. So, different signals are going in a different scale along the Y axis. So, you also need to consider if the protein is very very less abundant in your samples you should use more efficient antibody. If it is highly abundant you may not worry much. If it is really low abundant protein particularly phospho proteins very few copies will get phosphorylated than that total protein right. So, in that way we should be very cautious in choosing the antibody. So, all in all I would like to say that if one stop database is available for antibodies which are approved for RPPA approach will be a ideal situation. So, the community or the society is working towards that they have formed a consortium with the different labs and all the labs will share the data and they will they wanted to fit it in the computer. So, this data is accessible to open anybody then they can use the antibody information from which company which catalog number and so on then they can use for RPPA approach directly. So, this is now in the process, but another approach the protein at last company is included in their catalog of validation steps is by RPPA also. So, if you look at the data sheet of any antibody if you purchase then there will be different form of applications like compatible for western blotting, immunoestochemistry, immunofluorescence and all. So, this company included another validation point that it can be used for RPPA or not. So, if the single peak is there that means it is useful for RPPA approach. They also have the catalog indicating that whether it is go, no go or use with caution for RPPA approach this protein at last. So, next is a preparation of protein microarray. Here actually it is important to show you how to print and everything since I do not have instrument to print and show you all. So, definitely it is similar steps exactly same steps it follow as Sanjeeva explained in the morning the glass slide with a nitrocellulose membrane in white color and the sample is printed by a small pin from the machine. So, there are different methods are available to print. So, protein microarrays are typically prepared by immobilizing the proteins onto a microscope slide using a standard contact spotter or non contact spotters. So, there are different methods are available one is a robotic method which is a contact microarray method. Contact microarray method means needle will go and touch the membrane and disperse the liquid in that. So, that sample will not disperse in a big size of the spot it will accommodate sorry small area on the membrane will accommodate the larger quantity of sample. These are the remaining three methods are the non contact methods ink jet method piezoelectric spotting and photoliography. So, maybe many people know about the ink jet method it will spray the sample near to the glass slide. So, there is a chance of big wider spots than the concise spots. So, that you can accommodate less number of proteins on one slide compared to the contact now contact microarray methods. And also you can use different wide array of substrates, but in RPPA approach we need a matrix coated glass slides. So, this matrix can be anything glass directly or gold or nitrocellulose or hydrogel. So, every substrate or every surface has its own advantages and disadvantages. So, in this case so glass is moderate reactivity towards a substrate and gold is low nitrocellulose is moderate and hydrogel is moderate. So, compared to this nitrocellulose is better because it gives low background and low fluorescence while detection methods are applied. So, that is the reason most of the people are using nitrocellulose coated slides whom I know at least may be others are using hydrogel methods as well. So, surface absorptions very low with the glass gold is very low and these nitrocellulose and hydrogel are very high that is why I preferred to use nitrocellulose coated slides. So, that nitrocellulose coated slide look like a white layer on the glass slide. You might have done thin layer chromatography in your masters right. So, exactly this thin layer is formed by silica gel in the thin layer chromatography. In this case nitrocellulose coated coating will be there that is it that is the difference. Once we have a glass slide. So, then you can decide the array. One slide can accommodate 2000 samples. Will you wait until 2000 samples will be accumulated from different studies? Not necessary right. So, then you can design the array into the miniaturized fashion. It is quite easy and possible. These chambers are custom made in our lab now are available in the market. So, one pad means it is like this. So, you can use one antibody for the entire slide for 2000 samples. If you have only 1000 samples you can divide them into two groups. So, 1000 here and 1000 here you can use two antibodies on each slide. And if you have very less number of samples you can also divide the chamber into 16 different chambers glass slide into 16 different chambers. So, that you can put 16 antibodies in one glass slide with maybe couple of hundreds of samples. So, in that way you can miniaturize your assay. So, in this case I will show you the examples. Here are the two pads. Here are the three pads. Here are the four pads. You can see this is the same sample set with the two different antibodies. Same samples with three different antibodies. Same samples with four different antibodies. That is it. It is just like a kind of miniaturizing your array. You can custom made array. So, once you have the array printed your protein of depending on the protein of interest you wanted to analyze and you can add antibodies and then probe and further next steps to be processed. So, after primary assume that in the RPPA also now you printed you added the primary after blocking and washing and everything. Here also nitrocellulose membrane empty spaces need to be blocked by using the blocking buffer. We use Rockland buffer which is a low fluorescence and after blocking your primary antibody hybridized. Then the second after washing to remove the excess antibody excess primary antibody bound to the membrane then you will add the secondary antibody. While choosing the secondary antibody you should be also very cautious. So, this choosing the antibody for detection is very critical and the outcome of the RPPA is based on the sensitivity and specificity of the primary antibodies. So, you should worry about the sensitivity and specificity, but also the signal detection method since the samples are delivered as a very very tiny droplets. And no protein separation is possible in RPPA. At least in western blot. So, your proteins are separated into small different bands. It is like a partial purification way, but here we are not following any separation methods right. So, the protein of interest may be expressed at a very low level and their visualization requires signal amplification. So, if the abundancy of the protein is very less, if your primary antibody is also very weak then still you have a chance to correct it in the secondary antibody detection method. As I said. So, how we can do that we will see. Thiramide signal amplification method has been widely applied to increase the sensitivity of the reverse phase protein arrays and calorimetric and communal limitations detection methods have very very high limitations in terms of linearity for quantification of signals. See for example, to get into your mind that what is the linearity means, if the if you use the fluorescence method the detection limits between 10 to 50 microgram. If you use calorimetric or chemiluminescence method if the signal gets saturated after 25 microgram you cannot have a proper quantification between 10 to 50 microgram range. But infrared detection system will have a very nice linearity between lower to high level of high level abundancy of the proteins. The fluorescence based signal amplification methods have been improved introduced to avoid issues with the signal detection. I will explain you one method. So, there are different methods are available. So, are applicable to development of the protein microarrays. It is again non labeling and labeling. So, I do not want to get into explain more about the labeling and non labeling methods and differences between these two. You can simply go through here and you can already see that labeling methods have labeling methods have little less usefulness in RPP approach than the non labeling approach. So, nowadays some people are using surface plasmon resonance as well. So, how it is a signal amplification method? You can see that simple spot is there and at primary antibody and labeled secondary antibody is added then you got a signal output. So, if you do not see any signal in this approach still you have a way to get it out. This is called AMSA method which was developed by one of the PhD student in my postdoc supervisor's lab, Heiko Mansberger. AMSA means antibody mediated signal amplification and you have a spot here. You can clearly see that you can have a spot here. This is a protein of interest and first antibody is added. Once the primary antibody is detected let us say for example, assume that it is a rabbit. The secondary antibody will be added anti rabbit raised in a goat. Then again we will select the third antibody which is raised against the goat in rabbit. This is again. So, now imagine that one label molecule became two label molecules. One molecule of infrared flow is attached with the one round of secondary to secondary antibody will have two fluorophores there. Then again if you repeat this for 10 times 10 up to three or four antibodies five antibodies one protein target it attached to five different signaling molecules. Then obviously, your signal will be increased by five times. So, this reiteration step will increase how it is. For example, in this example, in this cartoon you can see that the first direct detection you do not see any spot here. In the amplified detection first round of amplification we can start we can look at some of the spots in the right corner. After two rounds to three rounds of amplification then we can see almost all the spots in all the blocks here. So, that means, we are increasing the binding of labeled molecule to the protein of interest by repeating the signal amplification methods. So, only thing is cleverly we have to choose the what kind of antibodies to be used in the cycle primary, secondary and tertiary, tertiary to quaternary. So, this is a kind of cycle we have to repeat it. So, this is a reference if you wanted to go through how it is done here and you can clearly see that this red line is shifted from x axis towards to top. So, this shift of signal indicates that sensitivity has been increased, the signal detection has been increased. So, that we can visualize or we can detect the low abundant proteins in a large pool of proteins in the lysate present. This is a way and also linearity you can clearly see that. For example, here you can see this is a length. If you use infrared detection system, you will have this much distance to detect from lower concentration to higher concentration. If you use some of the other methods, you will end up only in the between the middle of the graph here up to maybe half of the concentration of the protein can be detectable. I am trying to just get you into the imagination, not able to show you the how signals can be amplified and all. So, this is a way. Once we have the array, once we have the signals here, definitely we need a soft ways to analyze. Nowadays, RPPA pipe which is a kind of open source software has been available and some of the companies have developed soft special soft ways to analyze the RPPA data generated, but it will have always commercial soft ways will have a lot of limitations. You cannot play with the data according to your biological question to be asked. So, keeping that in mind. So, that time myself also involved, Heiko Mansberger with Ulrika Korflab developed a RPPA analyzer which is a open source platform deposited into the website then you can use. So, there are different steps available here. After image analysis, the data can be collected in the form of GPR files and you will also give the file sample description, what kind of samples you printed there and slight description, what kind of antibodies probed like a 10 or 20 different antibodies against a set of samples and then you process it, the process data will be here and then automatically software look for quality controls means whether blank signal is really blank signal, the signal is really signal, what is the difference between blank signal to the real signal and also it will calculate the signal to noise ratio and if necessary to drop some of the arrays it will suggest what to drop, what to include then we can proceed further. Then obviously, once we have the quality control check, we always try to print a pool of sample in a serial dilution method. So, then the serial dilution will tell that whether antibody is detected the serially diluted samples, the linearity is 0 and the r-value is at least above 0.9, mean 1 is always ideal which will never achieved. So, at least 9 is the cutoff. Once we have this, then we will go ahead with the analysis further like a box plots, time dependent and all. So, when you have the box plots like here, you will see that whether the protein is upregulated or downregulated and also you can understand the signaling dynamics. Let us say for example, your samples are collected for a period of time, 0 minute to 10 minutes time. So, from 0 minute to 10 minutes time how the protein expression is going up or going down, simply you can see that the first few minutes signal is going up then started going down. So, signal dynamics we can measure and also we can fit this information into mathematical model to make a kind of network or to make a platform or to devise a pathway, regulation over the period of time, how it is getting stimulated, activated or inactivated and so on. Then obviously, you can design or you can draw a heat map, you can see that what are the different cluster of proteins are altering among the proteins I tested whether there have any relation or no relation. So, I hope today you have learnt about the importance of each and every step involved in RPPA workflow. You have probably now got a good glimpse and understanding of how different buffers behave differently with different sample types, their optimization is of utmost importance. Dr. Omani also emphasized on how it is important to establish good quality antibodies. And I must say this is one of the major issues in the entire proteomics community or the life science community to have the good standard antibody availability. And that is where the human protein atlas project along with several commercial companies have really provided the very high quality reagents which could be used for many implications including the one which we are talking today about reverse phase protein arrays. Today you must have also learnt about different detection methods and how each method has its own advantages and disadvantages. Dr. Omani further discussed about AMSA in detail. In the next lecture we will continue this interaction and talk about how to analyze reverse phase protein arrays microarray based data set. Thank you very much.