Tips For Making Research Efficient

Insights from Priyamvada Jayaprakash. Connect at Her LinkedIn Profile. She’d love to discuss her PhD experience with you and get insights on opportunities related to tumor immunotherapy.

Challenges I Faced During my Ph.D

I love Biology

Biology has always fascinated me. Understanding how cells and their environment team up to create a functioning organism mesmerizes me. So, without a second thought, I decided to pursue my Bachelors in Biotechnology when I was a senior in high school. During my coursework, I gained an introduction to diverse areas spanning genetics, biochemistry, cell biology and immunology. I learned to appreciate not only the complexities of organisms at a molecular level but also the fact that some of these molecular underpinnings can be explored and manipulated for the betterment of people. Towards the end of my 4 years, I realized that my passion for science wasn’t quelled by my undergraduate education and I pursued my scientific journey by accepting a PhD position in the University of Southern California (USC), Los Angeles. My thesis was on understanding the importance of secreted heat shock proteins (Hsps), Hsp90α and Hsp90β, in wound healing and breast cancer metastasis.

Like any other PhD student, I spent the initial few months (and a lot of time in the future) reading and understanding “literature”. This meant a thorough knowledge and understanding of what findings have been accomplished in my research area- what ideas have worked and more importantly, not worked. Fellow researchers would definitely know “Pubmed”, an extremely useful database from NCBI (National Center for Biotechnology Information), that lets you search for papers of a specific area- all in one location. I cannot emphasize enough how much literature is important in the life of a PhD. It is especially important for a a naïve undergraduate like me from a foreign land, with no previous experience in a research lab.

Question. What are the downfalls of “literature search” in general vs. PubMed specifically? Or are they the same?
Priya: Searching in general gives a lot of redundant and irrelevant information such as random articles/blog posts etc, most of which are not helpful. But I feel Pubmed search is more streamlined to identify publications of interest. To the best of my knowledge, finding papers is either through Pubmed or a general Google search.

The Research Process is Inefficient

Hence, it came as a shock to me when I realized Pubmed is probably the only database that lets you search for papers in one go. More appalling was the lack of accessibility to all journals and papers one might need. Most journals charge an “article processing fee” for making research work “open access” and unfortunately, many labs are not financially equipped to pay such fees. As a result, research is not freely available to everyone in a timely manner. This is further exacerbated by the fact that the research environment is for the most part competitive, rather than collaborative. A number of my friends and myself have been instructed to be cautious while sharing interesting findings that were still unpublished. Conferences are a great resource of getting to know what’s out there, but most labs are skeptical about sharing their new findings in the fear of getting “scooped”. However, an ideal research environment is one where everyone focuses on asking the right scientific questions, identifies relevant ways to answer the questions, establishes productive collaborations and works toward the betterment of the society. We should create a forum where researchers are able to share their findings with an open mind and get ideas from fellow colleagues- our scientific progress can only be improved by engaging more scientific minds.

Question. This supports ResearchGate. Do you think it adequately solves your challenges? What’s missing?
Priya: Honestly, I need to use ResearchGate more to comment on it. But from what I know, more scientists need to be part of it and be made aware of it as well.

Funding Issues have changed Science

In the recent past, there has also been a rapid decline in funding. NIH funding is being allocated to projects that have a high probability of being a success. Hence “risky” projects have taken a back seat, which means innovation, the most important quality of a research, is at stake. I did my PhD in a lab that was hit by the funding decline. The lab worked at the junction of basic and translational research. We performed in vitro experiments and identified secreted Hsp90α as an important pro-motility factor. We then performed in vivo experiments in mice and pigs to prove the importance of the protein in wound healing and breast cancer metastasis. Compared to labs that worked on drug screening, my PhD lab, similar to most labs, focused on identifying something that could definitely benefit people, in the long run. Hence, I had to restrict my ideas to experiments that we could afford. The funding decline also meant that scientists were not open to trying out new things. Scientists feared any change and “played safe” with their ideas. The goal has now become “publish or perish”.

Question. What does this physically mean? Is your research stifled?
Priya: The funding decline did not affect access to literature. My experience is mainly from inability to buy the right reagents/chemicals required for research. Recombinant proteins and antibodies are expensive and sometimes, you prefer buying these only from specific companies due to the quality. Thus, in case of a funding decline, you either do not buy the reagents you need or you settle for one of less quality. Thus research does get stifled.

Question. What does it mean to “play safe” and not really expand scientific discovery?
Priya: I can comment on this from the perspective of a researcher working in cancer biology. Cancer is a complex and heterogenous disease and 1) multiple targets are required to treat the disease 2) multiple approaches to identify the targets are required. Thanks to modern technology, there are high throughput screening methods to simultaneously identify multiple targets specifically expressed by cancer cells. However, these methods are expensive to execute and many labs are unable cannot afford them. Thus, many target proteins are understudied, which means the number of “druggable” targets in cancer patients are being understudied.

From the point of view of a lab with low funding, identifying a single protein that is specifically expressed by cancer cells can fetch them a paper and therefore more funding. This highlights the “publish or perish” scenario, but does nothing to advance scientific contributions to cancer research. In other words, “playing safe” affects scientific progress globally.

Research Lacks the Right Tools

As a graduate student, there were a lot of things I learned, apart from the science. I spent a lot of time identifying the right reagents for my experiments. Any student who has done western blotting will know that many times antibodies either a) do not work, b) have a lot of background noise or c) have non-specific binding. Not all antibodies work for all purposes. Hence, antibodies from a specific company may not all be amazing. Even though I use literature as a starting point, it requires a lot of trial and error, which means a lot of time and effort on the my part. But, not many resources have helped with this. I used VWR as an online resource to purchase reagents. However, VWR is merely a database that lists different companies offering a specific product. It provides no information on how good the product is.

Hmm, this gets me thinking…I’m happy spending time on Amazon getting a case for my iPhone. Amazon gives me a table comparing different vendors, with specific features of each vendor, the associated product, the price and reviews of each. Why can’t there be an Amazon for science? It would make a huge difference to my professional life. I would highly appreciate a resource that does this and provide reference papers that have used the product. Similar to the star ratings we find on Amazon or eBay, it would be very helpful and time saving for researchers if there were a good rating system for life science reagents too.

Identifying the right protocol is a challenging task too. We need to conserve time and resources…it would be a great help if some online platform provided references to other researches that have already standardized the protocol, along with information on the reagents used.

Question. How do you currently search for protocols and products?
Priya: Currently, I use a general Google search to look for protocols and VWR for most of my purchases. The problem is mainly that I get a lot of redundant and irrelevant information. In addition, it is difficult to verify if the protocols have been standardized and come from a reputed lab or institution. Also, there’s no proof if the protocol has worked for them or not. Hence, if I have a visual evidence (aka reference papers), it is more convincing.

Conclusions and More Ideas

I consider myself very fortunate to have obtained admission into a prestigious graduate program in the United States. I came to the country with no prior experience. When I look back, I think that certain tools may greatly benefit many young students. One tool I can think of is a visual pathway explorer. Biological processes are dynamic in nature and I have always wanted resources that can teach me a specific biological process more visually. Currently, I use YouTube for this purpose, but it does not have videos for everything. I think it would be amazing to have an interactive tool where a pathway/process is taught illustratively and questions can be posted on the website, which will be addressed by experts. Imagine how many ideas can be sprung out of this simple idea!

Science is fascinating, but with challenges of failure and lack of accessibility to resources, it can get pretty frustrating too. Thus by providing the right accessibility to constructive collaborations, the right protocols, and the right reagents, the life of scientists can be more productive.

Question. If you wanted to make research more efficient. What kind of company would you make? What products would it have? Assuming you have an unlimited budget and/or time.
Priya: I would create a company that acts as a liaison between labs and service/reagent companies. For example, for my research, I would need diverse products ranging from a specific custom-made cell line, lentiviral knockdown systems, CRISPR/Cas9 reagents, specific transgenic mouse models or diverse services ranging from microarray analyses, RNA seq analysis etc. Diverse types of companies provide these reagents and services. For example, IDT is the company I use to generate my oligo constructs for cloning while I use Jackson labs for mouse models and Affymetrix for microarray services.

My company would be like a liaison that obtains the needs of specific lab (s) and get in touch with these diverse companies. Every lab can create a profile on the company website and use it as a single portal to add these diverse orders. My company would communicate with the different companies and provide the reagents & services to the labs. This will be the service wing of the company. In addition, my company will also provide analyses software for the different high throughput techniques at a subsidized rate for customers. If feasible, certain bioinformatic analyses might also be performed by my company for a subsidized rate.

In addition, my company will also have a scientific wing where I would have a board of expert scientists, postdoctoral researchers and other experts in different fields, for eg, neuroscience, immunology, proteomics etc. If a lab is confused about how to address a specific goal, they can post the questions via their profile on my company website and they will be addressed by the respective experts.

SciPrice and SciGine are Solving Challenges in Research

Many of the challenges that Priya has outlined are now being tackled by SciPrice and Scigine.

SciPrice is a marketplace for antibodies which focuses on providing all the information possible for each product. It’s an Amazon for Science.

Scigine provides protocols for Biology from reputed journals along with the materials that were used. In combination with SciPrice, both services help make research more efficient.

Thank you for reading!

Western Blot Theory and Method Guide

Western Blot Method Guide and Step by Step Procedure

Overview of Western Blot Method

A western blot enables sensitive detection of specific proteins from a solution containing multiple proteins. This is an essential biology technique and one of the cheapest methods that can be utilized to analyze proteins. To perform a western blot first separate proteins based on their mass and charge via gel electrophoresis, and then follow up by detecting the protein of choice with a specific antibody. Typically, researchers will use western blots to separate proteins from cell media or from cell lysates. For example, if you wanted to find out how much actin your cells are expressing, a western blot can easily compare actin amounts between different cell types. It’s also likely that you will be using western blots when producing proteins in mammalian and insect cells.

In a typical western blot procedure, cells will first be lysed and the amount of protein will be determined using a spectrophotometer. Then a gel will be made and the total protein from the cell lysate will be loaded into wells in the gel. After applying an electrical field, the proteins in the gel will begin to migrate down and separate into distinct bands based on the size and charge of the protein. After the smallest proteins reach the bottom of the gel, the electrophoresis will be stopped and all proteins on the gel will be transferred onto blotting paper so that they can easily be handled. Finally, antibodies that recognize the proteins of interest will be added and detected via chemiluminescence.

Here is a step by step illustration of how to perform a western blot:

Western Blot Scientific Method Guide

Western Blot Step By Step

SDS-PAGE Western Blot Step-by-Step Protocol

Western blotting can be used to examine the upregulation of RCAN1, a signaling molecule in neuronal cell types.

Materials for Western Blots:

  • Rabbit anti-RCAN1 antibody (#SAB2101967, Sigma-Aldrich)
  • SDS-PAGE gel (Criterion TGX precast Stain-free Any kD gel, #5678124, Bio-Rad)
  • TBS (20 mM TrisCl pH 7.6, 150 mM NaCl)
  • Running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS)
  • Transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.05% SDS)
  • 4X SDS-PAGE loading buffer (Laemmli’s sample buffer #1610747 Bio-Rad; add fresh dithiothreitol to 10 mg/ml on the day of experiment)
  • Transfer membrane (0.2 um polyvinylidene fluoride membrane, #03010040001 Roche)
  • Secondary antibody (donkey anti-rabbit horseradish peroxidase-conjugate; #711-035-152 Jackson ImmunoResearch)
  • Blocking buffer (5% skim milk powder in TBS with 0.1% Tween20)
  • Immun-Star WesternC Chemiluminescence reaction solutions (#170-5070 Bio-Rad)
  • Dual color ladder (#1610374 Bio-Rad)
  • Blotting paper (ProteanXL, #1703966 Bio-Rad)

Western Blot Experimental procedure:

  1. Unwrap precast gel and rinse wells three times with running buffer. Assemble gel in tank and fill with running buffer.*
  2. In an Eppendorf tube add protein sample (30 µg) to 10 µl 4X SDS-PAGE loading buffer and add water to a final volume of 40 µl.
  3. Heat samples to 95°C for 2 min and spin briefly to ensure contents are at the bottom of the tube
  4. Load gel with samples and include ladder in one lane.
  5. Run gel at 200V for 30 min.
  6. While gel is running, soak two pieces of blotting paper (cut to the same size as the gel) in transfer buffer (approx. 30 min). Activate transfer membrane (also cut to size) by dipping in methanol, then soak in transfer buffer for approx. 10 min.
  7. Remove gel from tank and place in transfer buffer.
  8. Assemble transfer “sandwich” by placing down soaked blotting paper, transfer membrane, gel and blotting paper onto open transfer cassette (Turbo Blot transfer unit; Bio-Rad). Use a glass rod to roll across the “sandwich” to remove any air bubbles.
  9. Close cassette and run in machine (standard minigel program for 30 min).
  10. Remove transfer membrane from cassette, taking care to snip one corner to ensure orientation.*
  11. Incubate transfer membrane in blocking buffer for 1 h at 4°C with rocking.
  12. Pour off blocking buffer and add diluted anti-RCAN1 antibody 1:200 in 5 ml TBS with 2.5% skim milk power and 0.05% Tween20. Incubate overnight at 4C with rocking.
  13. Wash membrane three times in TBS with 0.2% Tween20 at 4°C with rocking, for 10 min each time.
  14. Incubate with diluted secondary antibody 1:2500 in 5 ml TBS with 0.2% Tween20 at 4°C with rocking for 1 h.
  15. Wash membrane three times in TBS with 0.2% Tween20 at 4°C with rocking, for 10 min each time. Rinse membrane briefly in water.
  16. Mix 1 ml each of ECL reagents in a foil-wrapped tube and add to membrane for 5 min prior to imaging on ChemiDoc MP imager (Bio-Rad).

Procedural notes for this Western Blot Method:

  • This precast gel contains 18 lanes with a loading capacity of 10-40 ug protein in up to 30 ul per well
  • Small needle-point markings can be added to membrane in-line with color markers which reduce in intensity following subsequent incubation and washing steps.
  • To make sure you know which step you are on, cut the bottom right side of your gel after running the gel electrophoresis.
  • In this method, the protein is denatured prior to running on the gel. This is called SDS-PAGE. By denaturing, you ensure that the size and charge are all that matter, as opposed to native gel electrophoresis where the conformation of the protein also matters.
  • Blots can be regenerated (the antibodies that were used for probing can be removed) by using stripping buffer. However, blots can only be stripped a few times before they have too much background noise to be easily analyzed.

Applications of Western Blots on Scigine (Search Engine for Scientific Methods):

References:

NIH Western Blot Reference
Western Blotting by Kurien et al

Good Video References Related to Western Blots:

Tools for Scholarly Search & Research in Biology

Efficient scholarly research helps to reduce unnecessary experiments

Scholarly Research reduces unnecessary experimentation

Nobody wants to reinvent the wheel. Look at all of the pain staking work that goes into re-testing previous experiments and developing methods that have already been known for decades. By taking our time to define our problems, hypothesize our solutions,and search through literature prior to executing on our plans, we can dramatically improve the effectiveness of our research. In fact, people dedicate entire chunks of their lives to being good at searching and testing! (Hint…it’s called a PhD).

In this blog post, I wanted to discuss the tools that are available to help Biologists and biochemists perform effective research. In particular I wanted to focus on biology-related search engines and their development/features since their inception.

A list of tools for biologists to efficiently perform their scholarly searches

  1. Scigine: A search engine for methods in biology, biochemistry, pharmaceutics, and clinical science. Provides step-by-step methods along with the ability to take “notes“, modify methods, and view them on a phone/tablet while performing experiments. Users can also share their notes with colleagues with the click of a button. (~600,000+ methods)
  2. Protocols.io: A “git-hub” for biology with the ability to “fork” methods and modify them. Also includes the ability to form groups with others to share methods online.  (~700 methods)
  3. Protocols-online: A collection of methods aggregated from multiple authors along with forums to ask questions and trending jobs for biologists and biochemists. (~1000 methods)
  4. Vadlo: Or “Fig”, hones google in to specific biology-related websites by providing a custom search text box. It is possible to use this search feature to broadly find methods, presentations, and articles that would normally be found through google.
  5.  DOAJ: The directory of open access journals provides access to over 1.9 million journal articles in biology, biochemistry, and related fields. In case your library does not provide access to journal articles, the DOAJ is a solid resource that is freely available.
  6. PubMed: From the website – “PubMed comprises more than 26 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.” This is the go-to site for scholarly research in biology and related fields.
  7. Microsoft Research Tools: A collection of online tools for genomics and bioinformatics provided by the Microsoft Biology Initiative.
  8. PubAlert: Automatic weekly or daily digests of PubMed searches for your keywords. This is a great tool for keeping up with the latest research in your field. Example, setting up an alert for “Actin” will provide you with an email digest of the most recent articles with “Actin” in the title or abstract.

Hopefully, this blog post provides a good “lay of the land”.

Enjoy the list of tools!

Karthik

SciGine: Inception of A Biology Methods Search Engine

Biology and Biochemistry Methods Search EngineDoing a PhD is about the pursuit of knowledge

The typical biology PhD student spends 5+ years in a PhD program. If you were to follow a student, you might start in the morning by watching him/her looking through papers to check what latest research might be out there related to their topic. Have they been scooped? Is there contradictory research out there that they now have to mention in their next research paper? What new information can they glean from their PubMed searches to understand their research challenges better and inch their way forward with their existing hypothesis?

After performing broad searches on their field of specialization, they would then narrow down their focus on their task at hand. How do I get to my next publication? How can I prove that my protein is being expressed? Is it being degraded by proteases? What kind of data will I need to prove my hypothesis? Really, completing a PhD is all about answering questions and pursuing knowledge — ie performing Biology and Biochemistry ReSearch.  But, the tools that are typically used in this search are not efficient or complete. Academic research, even though it is considered at the edge of knowledge and understanding, moves at a snail’s pace because it lacks the right tools.

 

Improving research efficiency in Biology by using free tools like SciGine

This was the reason that SciGine was created. By using the SciGine search engine, I was hoping that future generations of Biologists and Biochemists would be able to more efficiently tackle their research. The website is set up such that any one can use it by going to SciGine and typing in the search box. The next page shows up results similar to how PubMed and Google organize results as individual biology methods with their search query highlighted along with the author name and time/year of publication. Clicking on an individual method then takes the user to a split screen view consisting of the method steps on the left and a notes section on the right.

I realized that many times, the methods we use for research are amalgamations from multiple articles. So why not design a user interface that allows visitors to take notes while viewing multiple methods easily? On a typical method such as Western Blotting for Kidney Amino-Acid Oxidase this makes method development very simple. I can search for multiple western blot methods using the Browse or Search functionality and then take notes on what exactly I want to do with a protein of interest to me on he right side of the screen. With over 1000+ results from a simple “Western Blot” search, I’m amazed at how much this would have helped me during my PhD.

Saving Biology Methods online for ease-of-access later in lab

Beyond simply taking notes, users can save their methods and protocols and keep a running list of them as part of their Online Methods Notebook. This page is for editing their user profile but also can be used as an online lab notebook. I can envision users using it to keep their day-to-day methods and results online or just to keep a general repository of the methods that they tried out. It’s especially useful to keep this information online because a user can, for example, share their immunoblot method with a lab mate easily and also view their method in lab via a phone or tablet while performing their experiment. There are also option to keep methods private (un-searchable) or public (searchable by everyone) so that important information doesn’t get into the wrong hands.

The ability to upload complete methods online also has the advantage of making it easy to write publications later on. Too often, in a publication, details of their methods are lacking.  By keeping track of what materials were used (especially their part numbers and vendors), the references that were combined to make a method, and how each material was used step-by-step, writing a “Materials and Methods” section for a publication will be a breeze. I want researchers to be able to determine if a chemical from Sigma Aldrich vs. Fisher Scientific was the culprit when it came to their bad data. SciGine will, I expect, improve the quality of research produced by it’s users.

Eventually, I expect there to be more viewers asking and answering questions specific to the methods that they find on SciGine. However, several other resources exist which can help with questions for now. In particular Research Gate and Protocols Online have active communities on their forums that make it easy to get questions answered.

Thank you for reading about the inception of SciGine and how it can be used to advance your Biology and Biochemistry research. Look forward to more posts in the future!

Sincerely,

Karthik