Equestrian Biochemist Heads to D.C.

I haven’t had the opportunity to touch base recently, but I wanted to take a minute to check in. A few updates about what’s been going on:

1. I submitted my dissertation to my committee (it’s 200 pages, this is apparently a popular question)

2. I scheduled my final examination (April 14th)

3. I successfully ordered my “Commencement Regalia” from the UK Bookstore during a SEC Tournament Game. Go CATS!

4. I interviewed for and landed my dream job as a Science Policy Fellow at the American Society for Biochemistry and Molecular Biology in Rockville, MD (D.C. Area)

5. I got a new puppy: Her Ladyship Chloe Noelle Spaulding of the Irish Setter Ranch

So, the new job means that I am moving to Washington, D.C. very soon. This also means that the hunt for a boarding facility and trainer for Garth pony has begun. Due to extreme cold, dissertation induced depression, and complete lack of time, Garth pony got to take off the last couple months from training. At first, this seemed like a horrible idea, but after about two weeks, everything changed about him. He stopped jigging going to and from his stall. He patiently came to the gate when he was called in from the field. He completely stopped trashing his stall at night. I’ve climbed on his back twice since his sabbatical began and each time he was a complete dream. Kind, willing, forward, and soft to the hand. I am so happy to I got to give him this time off, and I am eager to start training again now that Spring has arrived.

I have been looking for trainers in the D.C. area and have found some facilities at which I want to look.

The first thing that has shocked me about preparing for this move is that human housing costs A LOT MORE MONEY, but horse housing and training is VERY REASONABLE compared to Lexington! It seems that the area has such a wealth of highly qualified trainers that there is great competition in the market. (Win-Win for Garth Pony and myself!)

I’ll be following up with more blogs on all these subjects in the future and the whirlwind that is about to be my move to Maryland. Go CATS!

(Chloe)

So where does cancer come from?

I stopped by the sandwich shop at the Cancer Center today to grab my favorite Italian panini and a Cherry Coke Zero from the same tiny Filipino lady I have been purchasing them from for the last 4 1/2 years. As I was paying my bill, I mentioned to her that I was going to be defending my dissertation in April and after that I would graduate. She was delighted for me and told me as much. She asked what I was working on for my dissertation and what my degree was in. I explained to her that I work on trying to understand why some people become resistant to a certain type of chemotherapy and my degree would be in Biochemistry. Another woman who works in the little sandwich shop walked over and joined our chat.

She told me that her mother, father, and brother had each died of cancer: womb, brain, and lung, respectively (her words).

She asked me why I hadn’t found a cure for cancer yet. I tried to explain to her that as our understanding of cancer increases we realize that cancer is not one disease but an entire constellation of diseases that progress in similar ways but with unique molecular features. I tried to explain to her that we may never “cure” cancer but we will slowly chip away at cancer types and subtypes. We’ve already found therapies that can put some cancers at bay for years and even permanently, while other types persist, progress, and prevail. As I stumbled around my words and tried to give her an answer that would leave her both enlightened about the cancer research enterprise and hopeful about her own future, I realized that I probably wasn’t giving her the answer she was looking for.

Never one to shy from a challenge, I conveyed the importance of a healthy lifestyle (exercise, nutrition), the irrefutable effect of family history (the genetic factor), and that sometimes it’s just bad luck  who gets cancer (mutations).  Suddenly becoming very aware that I was leading a primer on the basics of cancer in the sandwich shop of the Markey Cancer Center with medical doctors and nurses and patients intently listening, I started to grab my sandwich and edge towards the door. As I was making my escape, my sandwich lady asked me, almost sheepishly, “where does cancer come from?”

This woman literally works in the Cancer Center at the University of Kentucky, and I was the first person she ever felt comfortable enough to ask this question to? Embarrassed or not, I walked back over to the sandwich counter and spent a few minutes with her. I explained to her that sometimes maybe just one or two cells in the body decide to grow when they are not supposed to. They grow and grow until they make a tumor, and when a few cells in that tumor decide they don’t like being part of that tumor anymore… they leave. They crawl away and find a new place in the body to grow, and it’s when they “metastasize” that it becomes really hard to fight them.

I’ve spent the last few months writing my dissertation, a book chapter review article, and a first author manuscript for a peer reviewed journal. I’ve written lengthy interest statements for jobs I want, and cover letters for jobs I may never get. I’ve given seminars, lab meetings, journal clubs. But today, I took the time to really talk to someone about what I do and why it matters to me and to them and to all of us. Climbing down from the ivory tower of academics seems like a terrifying descent, but what’s the point of investing in all this education if you can’t take the time to share it with someone? The next time my favorite sandwich lady wants to talk about science in the sandwich shop, y’all better pull up a chair, crack open your Cherry Coke Zero with me, and get ready for a biochemistry breakdown.

Applications and Revisions

Writing a dissertation and applying for science policy fellowships at the same time can leave you feeling a little bipolar as a graduate student…

I spend the entire day writing. I work on my dissertation for a while, take a break, work on fellowship applications, coffee time, etc., etc. In addition to the observation that it’s boring to just sit and write about one subject for endless hours on end, I feel like inter mixing them helps you to pass the day with out feeling too good or too bad about yourself.

Every time I submit my dissertation to my mentor, I admittedly receive it back with a glowing note that says “Wonderful effort,” or “The manuscript is blooming like a beautiful flower.” (Yes, my mentor really writes things like that, she’s Greek.) Unfortunately and not surprisingly, it is also dripping in red pen. Suggestions like: “Expand your discussion of the crystal structure of the Androgen Receptor” and “Do you really think the 10 pages I told you to write about the crystal structure of the Androgen Receptor are necessary?” Every time I sit down to work on this mammoth document, I feel sad, fairly bad about myself, and usually a little overwhelmed. Time to switch it up…

I grab a fresh cup of coffee and open up one of my fellowship applications. I get to write about the future and what I hope to accomplish. I get to think about things like science policy, communicating scientific concepts, and the importance of STEM education. I get to write about my great qualities in my letters of interest and highlight my achievements as I repeatedly revise my resume. By the time, I assemble my application packets with letters of recommendation in tow, I feel pretty darn excellent about myself, the future, the world.

…Beep Beep…. New Email.

---

Sarah,

I have more dissertation revisions for you ready in my office. Please come get them ASAP. Also, I thought of 27 more experiments we could do before you graduate. Let’s discuss.

Sincerely,

Greek Boss Lady

---

I am not done with my dissertation… yet

I was absolutely flabbergasted when I walked into the Markey Cancer Center this morning and with an absolute seriousness the medical coder on my floor asked me “Are you done with your dissertation, yet?”

After flying into a rage in which I told him I would never speak to him if he ever dared to ask me that question again, I have tried to collect myself to proceed with my work for the day. Cell culture, taking care of immune system deficient mice, trying to write as many pages of my dissertation as possible, new draft of paper to be submitted first of the year, ordering lab supplies, cleaning up after my lab mates, keeping up with science policy updates, taking care of my husband, horse, dogs, cookie baking, laundry, Christmas present wrapping… head desk. No, I am not done with my dissertation yet.

Caution: If you ask me “Are you done with your dissertation yet?” I will likely scream at you like I just did to the medical coder in Markey. Feel free to ask me “How is your dissertation coming along?” “Is writing your dissertation as challenging as you thought it would be?” or my personal favorite “What is your wine to pages of dissertation per day ratio?”

I am really trying my very best to get my dissertation done, I promise. I am writing 6-7 hours per day, but I still have to fit in all the other things I need to accomplish. Being a wife, daughter, PhD student, equestian, biochemist, dog mom, and maid is a challenge. It’s hard to remember which direction I am headed in all the time, but I’m doing my best and I’m making a lot of progress. So for now, that has to be enough because I am going to finish that dissertation, and when I do, I’m going to slam that behemoth thing down on the medical coder’s desk and say “I’m done, now!” Ok, that’s not true, I totally won’t do that. But be certain, that I will imagine doing that. I’m gonna imagine it so hard.

A Knight in White Lab Coat

I’ve been wrapping up my last experiments for the paper I have been working on lately. One experiment that I needed required the use of  flow cytometry (“flow”). This is really a very basic experiment and the original use of flow. You treat your cells with whatever agent you’re interested in, in my case a chemotherapeutic drug. Then you use a DNA binding dye to allow you to determine how much DNA is in cells. In my case, I expected their to be 2-6 times as much DNA inside the cells I treated, compared to normal because of the way this chemotherapy affects cells.

(Figure: from AbCam)

We have a really great core facility here at the University of Kentucky that specializes in flow and I have been annoyingly emailing back and forth to get protocols, ask advice, and prepare for my experiment. Finally, the cells were ready, the reagents had all arrived, and I was ready to perform the experiment. I followed my protocol to perfection, everything was going great, scientific euphoria was within my grasp and on the final step the wheels came off. The last step in the protocol was to utilize a cell strainer to strain the cells. I sat the strainer on top of the conical tubes, I gently pipetted my cell solution up and down, and then I transferred the solution to the strainer. I watched. I waited. The cell solution sat on top of the strainer. It stared blankly back at me indifferent to the amount of work I had put into collecting and preparing it. I tapped the strainer. I whacked the strainer. I tried pipeting the solution THROUGH the strainer. Nope. Time was ticking by and my appointment with the core facility had already started 15 minutes earlier. I called the core scientist and begged for more advice. Our conversation went something like this:

Me: “Hi there, this is Sarah. I am supposed to be there right now for my appointment, I am so sorry I am late but I can’t get the cell solution through the cell strainer. (This should be read with fear and panic in your voice)”

Core Scientist: “OH! SLAM it on the table. That happens all the time.”

Me: “Really? Are you sure?”

Core Scientist:”Yep, slam it.”

I hung up the phone and walked determinedly back to the bench. I picked up the first sample and looked at it in my hand.  I slammed it. Suddenly, the earth shifted, the heavens parted and the cell solution slipped silently through the strainer. I worked down the line of samples eagerly slamming each one down on the counter, then flipping the strainers off, capping the samples, grabbed my ice bucket  and ran out the lab door.

I sprinted across the Hospital to the room that housed the Flow Core facility. I was only 20 minutes late for my appointment and surely they could run four little samples for me before I ran out of time. I busted through the door triumphantly slamming my samples down on a desk only to realize that I was not in the Flow Core but inside some man’s office.

He looked at me, clearly very confused, and asked “May I help you?”

I started to explain to him that I was trying to get to the Flow Cytometry core facility, I was late for my appointment, and that the Exchange system stated it was located in this room. He kindly explained that it USED to be in that room but it had relocated quite some time ago, but he was not sure to what room. I snatched my ice bucket full of samples from his desk, and took off running back through the hospital to my lab.

Bounding through the door of my own lab, I went to my computer found the correct room number and took off again back across the hospital. As I skidded to a halt outside the Flow Core, I swallowed my pride and stepped inside. There were ten minutes left of my appointment. I explained my ridiculous series of events and the core scientist took pity on me. She told me she’d help me until the next appointment arrived, but then she had to stop. She worked quickly and had the machine set up to run my samples in only a few moments, when another scientist walked through the door with a slew of samples. My heart sank as we had yet to run a single sample for my experiment. At that moment, a Post Doc who was only in the Flow facility to observe and learn about another more sophisticated machine walked over and offered to run my samples for me. After briefly conferring, the Flow Scientist agreed that the Post Doc could run my samples if he wanted, and he agreed. He spent time showing me how the machine worked, how to read the settings and data readouts, and even showed me a funny poem about Flow Cytometry. We ran the samples and despite the calamity of the last hour, my data looked really great. The pilot experiment gave me really important information that I was able to use to design a full scale experiment.

After all that hullabaloo, I forgot to ask the Post Doc his name. So for now, I am just thankful for that Knight in a White Lab Coat who snatched my experiment back from the brink of utter failure and I’ll just wait for an opportunity to pay it forward.

Protocol: Western Blot

Western Blot

Reagents:

1. RIPA
2. Running Buffer
3. Transfer Buffer
4. Upper Gel Buffer
5. Lower Gel Buffer
6. PBS
7. TBST
8. Blocking Buffer

Plating Cells

1. Using T-75 Flasks, plate cells at consistent concentration across Flasks
   1. Cells should be plated such that they are nearly confluent.

Treating Cells

Cell Type:____________________       Time:______________________________

Treatments:

Treatment 1:______________________

Treatment 2:______________________

Treatment 3:______________________

Treatment 4:______________________

Treatment 5:______________________

Treatment 6:______________________

Day 3 Preparing Cell Lysates

1. Aspirate off media from well completely.
2. Wash with PBS or HBSS.
3. Trypsinize cells from T-75 Flasks or directly scrape off if using 60/100mm dishes.
4. Obtain cell pellet, centrifuge @ 2400 rpm for 10 min
5. Wash cells 3x in ice cold PBS & centrifuge @ 2400 rpm for 10 min
6. Add 400uL RIPA buffer (ice cold), volume dependent on size of cell pellet
7. Freeze thaw 3x on dry ice
8. Homogenize via repetitive passage thru 23 gage needle & 3mL syringe
9. Spin down @ max g 15 min @ 4 degree C
10. Collect supernatantà Cell lysates, store @ -20 C until Protein Concentration Determination

 

 

Determining Protein Concentration

1. Utilize BCA Protein Assay and Lab spectrophotometer
2. Calculate Reagent needed:
   1. Standard Curve: 1mL
   2. Each Sample: 400uL
3. Add 50uL BCA Reagent B to each 1mL of BCA Reagent A
4. Add 5uL of standards to each well of 96 well Plate
5. Add 1uL Protein extracts to each well of 96 well Plate
6. Add 200uL BCA Reagent Mix (A+B) to each well of 96 well plate
7. Incubate @ 37C for 30 mins
8. Read @ 595nm on Spectrophotometer plate reader
9. Standard Curve Calculation and determination of protein concentration

Preparing Samples for Electrophoresis

1. Add Sample buffer to cell lysates
2. Heat denature samples by boiling in H2O bath for 3-10 minutes and cool to RT before loading into gel

Preparing electrophoresis gel

1. Prepare resolving gel, adding ingredients in order listed

Resolving Gel Reagent: 12%	1 gel (mL)	2 gel (mL)
ddH2O	4.4	8.8
40% Acrylamide/Bis	3.0	6.0
Gel Buffer (Lower Gel)	2.5	5.0
10% SDS	0.1	0.2
10% APS	0.05	0.10
TEMED	5uL	10uL

2. Pipet resolving gel into the gel casting module.
3. Carefully, layer ddH2O or Saturated Butanol on top of resolving gel.
4. Incubate @ RT 20-30 mins, pour off ddH2O

 

 

 

5. Prepare Stacking gel.

Stacking Gel Reagent: 4%	1 gel (mL)	2 gel (mL)
ddH2O	3.2	6.4
40% Acrylamide/Bis	0.5	1.0
Gel Buffer (Upper Gel)	1.25	2.5
10% SDS	0.05	0.10
10% APS	25uL	50uL
TEMED	5uL	10uL

6. Select appropriate comb and rinse with ddH20, dry with Chem Wipe
7. Pipet Stacking gel over Resolving gel in gel casting module.
8. Apply comb, carefully checking for bubbles.
9. Wait 20-30mins, carefully remove comb.
10. Pipet ddH2O or Running buffer into wells to remove any “junk” from the crevice.

Assemble Electrophoresis Apparatus

 

 

Load Protein Samples into the Electrophoresis Apparatus

1. Load a consistent amount of protein not volume into the gel!!!!
2. Add Protein Ladder as a marker
3. If there are unused lanes of the gel, fill with 2xSDS Sample Buffer to prevent lanes running crooked
4. Load 30-60ug Protein / well

 

1	2	3	4	5	6	7	8	9	10

5. Run at 100V for 1-2 hrs , Watch your protein ladder and loading dye for ideal protein placement on gel.

 

Protein Transfer

1. Prepare Transfer Buffer: See reagents.
2. For 1 gel: 1 nitrocellulose PVDF membrane, 2 Blotter papers, 2 sponges, and 1 sandwich cassette
3. Lay Sandwich cassette down in Transfer Buffer Black Side Down
4. Equilibrate membrane in 1oo% methanol
5. Equilibrate everything else in Transfer Buffer
6. Lay down
   1. 1 sponge
   2. 1 filter paper
   3. 1 gel
   4. 1membrane
   5. 1 filter paper
   6. 1 sponge
7. Close the sandwich and load into transfer apparatus, black side to black side.
8. Place ice tray into transfer apparatus if needed and fill to top with Transfer Buffer.
9. Run in cold room or on ice
10. 2 Run Time and Power Options:
    1. Overnight @ 45V
    2. 1-2 Hours @ 400mAmp

Blocking the Membrane

1. Prepare Block: 1.25g Dried Milk + 25mL TBST
2. Block overnight @ 4 C or 45 mins on Shaker @ RT

 

 

Antibody Incubations

1. Incubate the Primary Antibody with the membrane
2. Starting Dilution 1:2000, overnight @ 4 C
3. Remove the membrane from 1’ Ab and wash in TBST, 3x 10 mins
4. Incubate the Secondary Antibody with the membrane
5. Place on rotating platform for 1-2 hrs @ RT
6. Wash in TBST 3x 10 mins

Revealing the Western Signal

1. Prepare ECL Mix
   1. Mix detection solutions A & B in a ratio of 40:1
      1. E.g. 2mL soln A + 50uL soln B
   2. Drain excess wash buffer from the membrane and place protein side up on a sheet of saran wrap.
   3. Pipet solution onto the membrane
   4. Incubate 1min @ RT in dark room
   5. Blot excess detection solution off and process immediately.
2. Expose top and bottom of film for 10s, 1 min and 5min. Process film in second floor film processor and expose subsequent film accordingly.

 

 

Reagents:

1. 5x Running Buffer
   1. Dissolve in 700mL ddH2O
   2. 1 g Tris Base
   3. 94g Glycine
   4. 50mL 10% SDS
   5. BTVol @ 1L with ddH2O
2. 1x Running Buffer
   1. 200mL 5x Running Buffer
   2. 800mL ddH2O
3. 10x Transfer Buffer
   1. 30g Tris Base
   2. 144g Glycine
   3. 100mL 10% SDS
   4. BTVol @ 1L with ddH2O
4. 1x Transfer Buffer
   1. 100mL 10x Transfer Buffer
   2. 200mL Methanol
   3. 700mL ddH2O
   4. Store @ 4 degree C
5. 4x Lower Gel Buffer
   2. 8g Tris Base
   3. ddH2O to 300mL, adjust pH to 8.8
   4. 6g SDS
   5. ddH2O BTVol. @ 400mL
   6. Store @ 4 degree C
6. 4x Upper Gel Buffer (Stacking Buffer)
   6. Dissolve 6.05g Tris Base in 40mL ddH2O
   7. Adjust pH to 6.8
   8. To 100mL with ddH2O
   9. Add 0.4g SDS
   10. Store @ 4 degree C
7. 2x SDS Loading Buffer: Sample Buffer
   1. Add to 15mL tube
   2. 25mL 1M Tris HCl, pH 6.8
   3. 0mL Glycerol
   4. 4g SDS
   5. 75mL of 0.04% Bromphenol blue solution
   6. Mix well and store @ RT
   7. Add β-mercaptoethanol fresh every time a protein gel is run
      1. 1.5mL 2xSDS Loading Buffer + 140uL β-ME per aliquot
      2. Store @ 4 C
   8. Blocking Buffer:
      1. Prepare Block: 1.25g Dried Milk + 25mL TBST
   9. RIPA:
      1. Take 10x RIPA buffer stock and dilute with ddH2O
      2. Add PMSF
         1. 6ul/1mL 10mM PMSF

Protocol: Subcellular Fractionation (Kit)

Materials Required:

1. NE-PER Nuclear and Cytoplasmic Extraction Reagents: 78835, Thermo Scientfic
   1. CERI
   2. CERII
   3. NER
2. Protease Inhibitors
3. PBS
4. Chilled Micro- centrifuge Tubes

Experimental Design:

Cell Line:___________________________________ Time:______________________________________

Treatment 1:________________________________ Treatment 5:_______________________________

Treatment 2:________________________________ Treatment 6:_______________________________

Treatment 3:________________________________ Treatment 7:_______________________________

Treatment 4:________________________________ Treatment 8:_______________________________

Protocol: (For adherent cell culture preparations)

1. For adherent cells, harvest with Trypsin and then centrifuge at 500 x g for 5 minutes.
2. Wash cells by suspending the cell pellet with PBS
3. Count Cells: # Cells / mL:__________________________________________________________
   1. Rule of Thumb: 2×10⁶ HeLa cells = 20uL PCV
4. Transfer 1-10 x 10⁶ cells to a 1.5 mL microcentrifuge tube and pellet by centrifugation at 500 x g for 2-3 minutes.
5. Use a pipette to carefully remove and discard the supernatant, leaving the cell pellet as dry as possible.
6. Add ice-cold CERI to the cell pellet (per table 1). Proceed to cytoplasmic and nuclear protein extraction, using reagent volumes indicated in Table 1.

Packed cell Volume (uL)	CERI (uL)	CERII (uL)	NER (uL)
10	100	5.5	25
20	200	11	50
50	500	27.5	125
100	1000	55	250

7. Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell pellet. Incubate the tube on ice for 10 minutes.
8. Add ice-cold CERII to the tube.
9. Vortex the tube for 5 seconds on the highest setting. Incubate the tube on ice for 1 minute.
10. Vortex the tube for 5 seconds on the highest setting. Centrifuge the tube for 5 minutes at maximum speed in a microcentrifuge (~16,000 x g).
11. Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube. Place this tube on ice until use of storage.
12. Suspend the insoluble pellet fraction produced in Step 4, which contains nuclei, in ice cold NER.
13. Vortex on the highest setting for 15 seconds. Place the sample on ice and continue vortexing for 15 seconds every 10 minutes for 40 minutes.
14. Centrifuge the tube at maximum speed (~16,000 x g) in a microcentrifuge for 10 minutes.
15. Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube. Place on ice.
16. Store extracts at -80C until use.

 

 

Protocol: Tips and Tricks of Culturing Select Prostate Cancer Cells

Cell Line	Culture Vessel	Media	mL	Moday	Tuesday	Wednesday	Split?
VCaP	T25	DMEM	6-8mL	x	x	x	1:2or3
LNCaP-FGC	T75	RPMI	12mL	x	x	x	1:3
PC3v567es	T75	RPMI	12mL	x	x	x	1:10
PC3v12	T75	RPMI	12mL	x	x	x	1:20
22Rv1	T75	RPMI	12mL	x	x	x	1:5or6

 

On top of every culture vessel is written the name of the cell line, the type of media it uses and the date it was plated.

I have tried to split all cell lines such that they won’t need to be split but cell lines which express AR can’t be split TOO low or they will die. (1:3 – 1:6 is the maximum they can handle).

Notes about important cell lines:

1. LNCaP FGC- these are very delicate cells. Suction media off very gently and use a slow setting when applying fresh media. Otherwise they will detach and die.
   1. When trypsinining this cell line, add Trypsin and leave the cells in the hood for 2-3 minutes. These cells do not need to be returned to the incubator for Trypsin because they are so lightly attached to the flask.
2. 22Rv1- These cells release vesicles into their media. This may appear to look like contamination, but if the media does not become “milky” they are just doing their normal thing.
3. VCaP-
   1. These cells act SUPER weird. They may form prostaspheres in the media and appear to be contaminated with round space ships. That’s their normal weird thing that they do. Just keep feeding them.
   2. When changing the media, do not remove all of the old media. Leave ~1mL of old media in the flask and add new media to the cells. They like their growth factors and cytokines to be around all the time. It makes them feel happy.
   3. These cells must be 100-150% confluent before splitting them. DO NOT SPLIT THEM until they are at least 100% confluent.

 

When splitting cells do not use HBSS (Hanks) to neutralize media after trypsinization. Use regular media in place of Hanks at all steps.

Protocol: Immuno-precipitation (Dyna Beads Kit)

Immuno-precipitation Kit Dynabeads Protein G

Kit Contents

Kit Contents	Volume
Dynabeads Protein G	2mL
Ab Binding & Washing Buffer	16mL
Washing Buffer	28mL
Elution Buffer	1mL

 

Required Materials

PBS w/ Tween 20

Magnet

Mixer (allowing tilting and rotation of tubes)

Cell Lysis Buffer

SDS-PAGE sample buffer

General Guidelines

Dynabeads Protein G have a binding capacity of approximately 8ug human IgG per mg beads.

I want to IP:___________________________________________________________________________

So I can IB:____________________________________________________________________________

Protocol

Prepare Dynabeads

1. Resuspend Dynabeads in the vial (vortex>30 sec or tilt and rotate 5 min)
2. Transfer 50uL (1.5mg) Dynabeads to a tube.
3. Place the tube on the magnet to separate the beads from the solution and remove the supernatant.
4. Remove the tube from the magnet

Bind Antibody

1. Add your antibody (Ab) typically 1-10ug) diluted in 200uL PBS with Tween-20 added to the tube from step 4 above. The optimal amount of Ab needed depends on the individual antibody used.
   1. IP Ab:____________________________________ uL:____________________________
2. Incubate with rotation for 10 min at RT.
3. Place the tube on the magnet and remove the supernatant.
4. Remove the tube from the magnet and resuspend the beads-Ab complex in 200uL PBS with Tween-20. Wash by gentle pipetting.

Immunoprecipitation Target Antigen

Antigen:____________________________________________

1. Place the tube containing Dynabeads-Ab complex on the magnet and remove supernatant.
2. Add your sample containing the antigen (Ag) (typically 100-1000uL) and gently pipette to resuspend the Dynabeads-Ab Complex.
3. Incubate with rotation or 10min at room temperature to allow antigen to bind to the Dynabeads-Ab complex.
4. Place the tube on the magnet. Transfer the supernatant to a clean tube for further analysis if desired.
5. Wash the dynabeads-Ab-antigen complex 3 times using 200uL washing buffer for each wash. Separate on the magnet between each wash, remove supernatant, and resuspend by gentle pipetting.
6. Resuspend Dynabeads-Ab-antigen complex in 100uL washing buffer and transfer the bead suspension to a clean tube. This is recommended to avoid co-elution of proteins bound to the tube wall.

Elute Target Antigen (Denaturing Elution)

1. Place the tube containing Dynabeads-Ab-Ag complex on the magnet and remove the supernatant.
2. Add 20uL Elution Buffer and 20uL 2xSDS (Blue Dye Stuff).
3. Gently pipette to resuspend the Dynabeads-Ab-Ag complex.
4. Heat for 10min at 90C.
5. Place the tube on the magnet and load the supernatant/sample onto a gel.

 

Protocol: Immuno-precipitation (Non-kit)

Immuno-precipitation

Reagents:
1. IP Cell Lysis Buffer + Protease Inhibitors (complete, Mini Protease inhibitor cocktail tablets) and PMSF

2. 1 x PBS (ice cold)

3. Protein G Plus/ Protein A Agarose Suspension

4. Trypsin (Optional)

5. 2x SDS Loading Buffer

Protocol:

Cell Line:____________________________  Number of Flasks Required:__________________________

Treatments: 1:_________________ 2:__________________ 3:___________________

4:___________________5:____________________6:__________________________

1. Plate cells to confluent, treat appropriately with interests of experiment, wash cells with ice cold PBS, &:
   1. Trypsinize, spin down to get cell pellet in micro-centrifuge tube.
      1. Wash 3x’s with Ice cold PBS 1. □ 2. □ 3. □
   2. Add IP Cell Lysis Buffer (200-250uL per sample)

OR

1. Add ice cold IP Lysis Buffer to the cells according to the table below and incubate on ice for 5 minutes with periodic mixing

Plate Size/Surface Area	Volume of IP Lysis Buffer
100×100	500-1000uL
100×60	250-500uL
6 well plate	200-400uL per well
24 well plate	100-200uL per well

 

2. Incubate on ice for 15 minutes, Pass samples through a 23 guage needle 7 times.
3. Spin samples for 15 minutes @ 4°C at Full Speed.
4. Plug in the rotator in the cold room and start rotating.
5. Transfer supernatant to fresh ice cold tubes and Perform BCA assay on supernatant collected after spin as per normal technique.
   1. For each ip you should use the same amount of starting protein. Generally, use as much protein as you can.  Typically, you’ll want a couple hundred micrograms protein for each ip.  Dilute each lysate for ip to 400 mL in lysis buffer with protease and phosphatase inhibitors.  Set 50 ug lysate aside to use as a whole cell control.
   2. 500-1000ug Protein / IP required, must use a consistent amount of protein across treatments for comparison purposes.

Sample 1: ug/uL:_______________________  uL for 500ug Protein__________________

Sample 2: ug/uL:_______________________  uL for 500ug Protein__________________

Sample 3: ug/uL:_______________________  uL for 500ug Protein__________________

Sample 4: ug/uL:_______________________  uL for 500ug Protein__________________

Sample 5: ug/uL:_______________________  uL for 500ug Protein__________________

Sample 6: ug/uL:_______________________  uL for 500ug Protein__________________

 

6. Pre Clearing: Optional
   1. Take 200 uL cell lysate and add (20uL of 50% Slurry)Protein G/A Agarose Beads
   2. Incubate @ 4°C for 30-60 minutes
   3. Spin for 10 minutes at 4° Transfer Supernatant to Fresh Tube
   4. Proceed to Step 1 of IP
7. Add Primary Antibody [1:200] (General Rule) to Cell Lysate
   1. Mix thoroughly with pipet and put on Rotator in Cold Room for 2 hours
   2. Primary Antibody Used for Pulldown: _____________________________
   3. Concentration of Primary Used:__________________________________
8. Add 15uL Protein G Plus/ Protein A Agarose Suspension / 1ug Primary Antibody
   1. ______________________ ug Primary Ab = _______________________ uL ProteinG/A
   2. Put on rotator in cold room for 1 hr
9. Wash with IP Cell Lysis Buffer: Spin down MCT contents @ 1-2 x 1000 rpm for 2 minutes
   1. Wash with IP Cell Lysis Buffer 3x’s □ 2. □ 3. □
10. Suction off all excess liquid, and then jam the pipet tip to the bottom of the MCT and remove all liquid in the beads as well.
11. Add 25 uL IP Cell Lysis Buffer back to the beads
12. Add 25 uL 2x SDS Loading Buffer and mix gently with pipet
13. Heat tubes @ 95-100°C for 5 minutes
14. Allow samples to cool and load in SDS PAGE gel for electrophoresis and western blot.