Teaching Points: Protein Purificatin Lab

VDS Staff,

The notebook for this one is due until on FRIDAY (unless they do the lab on Sunday – then it is due  Sunday night)

No lab report due for this one until after the Next wet lab (Characterization) – all they have to do is Post their Images from the lab to the Wikispaces.

There are 2 parts to the lab

PART 1: Purification

PART2: Nanodrop to measure amount of protein (this does not have to be done at the same time – but if the Nanodrop is available, they should do it)

The first steps are incubation steps that take awhile (so – to save time – they should immediately start these and then fill in their lab notebook and do Pre-lab calculations)

– while they are waiting they can 1. do pre lab calculations  2.  download the Journal Paper and look up the Maximum Wavelength for the last part of the lab

REAGENTS:

Don’t let them use TOO much Ni-NTA resin – it is expensive.

Don’t let them set the Benzonase out too long (must keep on ice)

For the Flow Through and Wash steps – they can use the ‘really cheap’, non-sterile tubes that we used for the Beer’s law lab. They should tape to label these – since they can be washed and re-used.

However, for the Elution 1 and 2 – they should used ‘fancy’ purple screw cap tubes

IMPORTANT CONCEPTS & SKILLS:

Emphasize keeping the protein and re-agents cold because the protein could denature.

What the lysozyme does

What the Benzonase does

Our protein is soluble and is found in the soluble fraction after clarifying the lysate

(from the cytosol – not a membrane bound protein)

The HIS tag on our protein binds to the Nickel on the beads (resin = beads = matrix)

The HIS tag is not ‘natural’ – it was put on their by genetic modification

What imidazole does. It competes with the Histidines of the HIS tag for the binding with Nickel. If you add enough imidazole – the protein will come off the beads.

TROUBLESHOOTING:

If there Elution 1 doesn’t come out purple – they should re-incubate their Flow Through and Wash with the Ni-NTA and let it run through again.

 

 

 

PTP1b spun down

The PTP1b in pNIC-Bsa4 in BL21(DE3) that leighanna grew up in 1 Liter was spun down and resuspended in ~11ml of Lysis Buffer.

Stored in -2odegC

Sample taken:  ~#1.5 after Lysis buffer added (100 ul)

Pellet weight: 6.5 grams for both combined (this seems pretty good)

Dr B

Mentor Tasks

Make plasmid stocks:

Grow up 2×80 ml of pGBR22 in DHFalphas overnight for MaxiPrep

Grow up 2×80 ml of PTP1b in pNIC-Bsa4 in DHFalphas overnight for MaxiPrep

 

Prepare PTP1b enzyme for new enzyme assay Lab:       (IN TEAMS OF 2)

Radhika: re-transform PTP1b into Bl21(DE3) and DH5alphas

Prepare 4 liters of LB  – autoclave

Autoclave big flasks – maybe use Biotech lab’s

Expression (overnight and then next day with IPTG)

Purification

Characterization

Concentration, buffer exchange

FPLC purification (Dr. B and Leighanna)

 

Once we have enzyme: PTP1b enzyme assay test

Also, GOLD Virtual Screen 3  Lab for PTP1b (using GOLD GUI to set up protein)

M&M for Expression, Purification, and Characterization

TUESDAY:  M&M for Expression, Purification and Characterization will be due

upload this to the Google Docs as a PDF in the

Results&Images/ReportonTarget/MaterialsandMethods Folder

Name it:   UTEID_YourName_Date_ MaterialsandMethods_ExpressionEtc.pdf

Inhibition Assays

VDSers – the inhibition assay protocols are posted. Yeah – the last experiments!

You should complete the other Enzyme Assays (vary enzyme, vary substrate, vary DMSO) before proceeding to the inhibitors.If you are still working on protein characterization – I would strongly urge you to being your assays and come back to the characterization steps.

Also, you show your enzyme assay results to Dr. B (electronically – blog preferably) before proceeding to inhibition.

The inhibitors are expensive and are common stock (i.e. we will all share them). So, it is important that these be diluted properly. Also the Inhibition protocol is a little different in that it requires a pre-incubation.

Images from the 11/2 Week

Fig. 1 - OD Valued Measured from Elution I after Protein Purification on 11/2/10. The OD Valued was .135 and the protein concentration is .13 mg/ml.

Fig. 2 - Elution I OD Value of .197 with .20 mg/ml. This is from Protein Purification on 10/29/10.

Fig. 3 - 2nd OD Valued Measured from Elution I after Protein Purification on 11/2/10. The OD Valued was .132 and the protein concentration is .13 mg/ml.

I was a bit confused by Fig. 2 because I actually obtained a solid OD value from this reading, but when I ran the Protein Gel, there was not a single band that showed up for Elution I done on 10/29/10. I took the sample on 10/29/10, but there should still be protein in the solution unless it was not mixed properly.

Protein Expression + Purification + SDS Page Gel

 

Fig. 1 - Protein Marker #SM0671 Lot: 00061923

Fig. 2- The SDS Page Protein Gel in 1X TGS Buffer Stained with an Imperial Protein Stain. Lane 1 – Protein Marker Lane 2- Protein Purification Waste Lane 3- Protein Purification Wash Lane 4- Elution I (11/2/10) Lane 5- Elution II(11/2/10) Lane 6- Elution I from Protein Purification on 10/29/10 Lane 7- Elution II from Protein Purifcaiton on 10/29/10 Lane 8 Sample #2 (Soluble Fraction after Sonication) from Protein Purication on 11/2/10

This week I had to do another protein expression on Monday and Purification on Tues, It was really quick. This time I wanted to do things different and actually take time to babysit my growth to .6 but again I had to go to class. When I got back to add my IPTG my growth was 1.125 and 1.197 for flask A and B with the LB and my CA7 BL21 bacteria cells respectively.  I really believe that adding the IPTG at .6 is the key component to obtaining an excellent yield from purification.  The CA7 Protein I have must have the best function at that growth number and missing that number is critical for the bacteria to begin making more CA7. After Purification, I worked on the SDS Page Gel/Protein Characterization. I realized that in my elution from 10/29/10, I had a low protein yield. When I did protein expression and purification again, I obtained a higher yield, but it was not excellent. The next step is to protein concentrate to get all the other proteins that are in the elution I from the second expression and purification. Calculations from Protein Purification will be updated on the blog sometime this week as well. After that enzymes assays will occur and I am crossing my fingers hoping that my protein is functional or back to expression. There is nothing much to rehash as I have done this process before last week. The only thing that was sort of new was the page gel, but the class did that last year. Once I began the gel, everything began to flow back in my mind. Oh! I pHed the Elution Buffer for everyone. Yeah! Also, this week I ran 8 GOLD Libraries. The Images and data form the aggregate is on Google Docs, but the libraries that are on the aggregate are the CB306, ChemBridge, KINA, LOPAC and ZINCL Libraries. I had to use multiple blades and uses mulitple processes on that blade just to get all of these libraries run. I had 5 libraries from the CAII Protein, but after cloning and a new protein I had to change my GOLD protein and start all the way over. Hopefully I have enough functional protein and a functional enzymes that I can do some testing.

Lysozyme

Due to the syringe fiasco, I had to redo protein expression. I used the shaking incubator in the biotech lab because ours had malfunctioned. I also got to use their fancy OD-o-meter. 🙂

I was able to get one of my concentrations to 0.719, but the other one was 0.830. In addition, I forgot to collect Samples 0 & 1.

Dr. B said that people were having trouble breaking the cell walls of their bacteria and expressing their protein, so we made some changes to the protocol. After centrifugation on Day 3, I added lysis buffer as well as 250 ul of lysozyme to my 15 ml tube. I stored the tube in the -20 degrees Celsius freezer, and the next day I sonicated my sample with an extra 1 x 30 sec at 90% cycle. Unfortunately, the sample was still stringy so the cell walls hadn’t been broken down as much as we would have liked. I then centrifuged my sample as stated in the original protocol.

After centrifugation, I noticed a difference in the appearance of my cell pellet. The first time I had done protein expression, my pellet had the texture of wet sand. This time, my pellet was less compact and looked like a piece of chewed gum. In addition, it was harder to pipette out the soluble fraction because the pellet was still kind of stringy. The changed appearance of the cell pellet was probably due to the lysozyme and additional sonication cycle.

Protein Expression

This last week I worked on protein expression. On day 3, our lovely mentors (THANK YOU KATHRYN! AND ADAM!) slaved away by OD-ing a bunch of people’s bacterial cultures, saving us a good 2 hours and 45 minutes in lab. Unfortunately, my concentration was much lower than everybody else’s (0.296 at 3:15 p.m.). I kind of expected my concentration to be low as there was a bit of a mix-up the day before with sharing and pouring everybody’s samples (did Damarcus do this step with us? Is this why his CA2 turned into CA7??) and not enough LB!

Later that day, I arrived at lab around 6:30 p.m. Thao informed me of my low concentration, and we went to the biotech lab with Dr. B. Nobody knew at the time whether IPTG had been added to my flask (it hadn’t – my fault – I should’ve come earlier), and since my concentration was so low, we decided to continue on with the protocol and centrifuge it.

The next day, I resuspended, sonicated, and centrifuged my sample. I also grew up another batch of protein in the shaking incubator for the weekend.

I came in on Saturday and OD’d my samples. Everything went smoothly, except I FORGOT TO SAVE MY SAMPLES and smashed a 2L flask in the sink.

Question

When I do protein purification again do I add more Ni-NTA (1ml) to the supernatant after sonication or do I just pour the solution right in the column and allow it to sit for 45 minutes?

What a Week- RE Digest, Protein Expression, Purification

Let’s Travel all the way back to 10/22/10. After I performed RE Digest for the second time, I had an epiphany. I have a new enzyme, CA7, so the RE Enzymes used from CAII probably will not work on my new protein. Let’s Check. Yes, it does not cut in the manner it would on CAII.

Lane I and II are skipped. Lane III and IV are the 1 kb and 100bp DNA Ladders respectively. Lane V is the uncut control plasmid of pNIC-Bsa4 with the stuffer fragment. Lane VI- is the DNA from Cloning of Colony 3 on the Master DH5alpha master plate. Lane VII- is the DNA from Cloning of Colony 7 on the Master DH5alpha master plate.

This was reassuring because I beginning to believe I was doing RE Digest improperly. Well after this, I sent off the sequence from Cloning of Colony 7 and Colony 4 to the ICMB Core, because Colony 7 resulted in four N’s for its whole sequence when the pLIC primers were used. While waiting on the results to come back, I put the DNA from Colony 3 and Colony7 in BL21 expression vectors and they came out great! I should have taken a picture of them, for everyone to see. Colony 7 looked weird again. The plate from Colony 7’s DNA from Cloning did not output  distinct colonies as Colony 3 did. Now, I did add all 25 ul of the Transformation solution to the agar plate, so it could have been overcrowding in Colony 7. After this was complete, I began this week looking at the DNA sequences obtained from the core. Colony 4 came out again with CA7, but for Colony 7 from Cloning, the core gave me a sequence with 4 N’s. I am beginning to believe that Colony 7 from Cloning has the CA7 DNA in solution, but not in the pNIC-Bsa4 Vector.  I choose Colony 3 to grow up my overnight culture, and then placed them in the 2 500 ml LB Broth for my OD Readings.  At 600 nm,  at 759 am the ABS value was .098 for B, and at 8:10 the ABS value was .110 for A. I was not able to babysit and check the OD values properly because I has class during the time of OD value check. After 2 hours, I came to check the OD values again and the 1st OD Value (ABS at 600 nm) at 11:30 was 1.606 for A and for B it was .856. (A and B come from the 2 500 ml flask that the solution of LB and bacteria were in.) After this, I added my IPTG and allowed it to incubate for 4 hours. After about 30 minutes, I realized I did not add my Kan to my new LB Solution, so I went on and added the Kan. Once that was finished, I put the LB in two tubes to allow them to centrifuge 20 min at 6000 g. The pellets weighed 48.17 grams. The pellets were resuspended and sonicated 2 days after that. Some of the solution spilled during sonication. After sonication the proteins were centrifuged again and then on to protein purification. The most tricky part of protein purification was the  balancing the ph correctly because the HCL and NaOH are so concentration and a drop could totally change the pH of the protein. Question: Were my protein’s denatured? My pH went down to around 5.2 went I added the HCL, could that have been the source of the low protein yield that was to come after purification. Once the pH was at the proper value, I added the 1ml of the Ni-NTA slush to the solution and allowed it to incubate for 45 minutes. Another issues that could have arose was that I did not incubate as often as I should have to allow the protein to bind to the Nickel. Next Time, I will put the ice bucket directly in front of  me and invert it every 5-10 minutes, so the Nickel will stick to the protein. I performed the rest of the task of protein purification and when the elution were taken I used the buffer that had 150 mM of NaCl instead of the 300 mM NaCl. 150 mM is close the the physiological concentration, but 300 mM creates the best protein binding environment to the Nickel.  Once purification was complete, I Nano-dropped the solution to find out my protein yield. The yield for elution one was .110 at the highest. I forgot to keep the picture of the graph for that because I Nano-dropped again an kept keeping positive and negative protein concentrations. So I am back to growing up my bacteria for expression. I went into lab today to make LB and autoclave it, but the autoclave was having issues. The solutions of LB are made, but they have not been autoclaved. When I do expression the next time I have to be more careful of what I am doing and make sure I have time to babysit the LB to grow to the proper value of .6.

 

All go for expression!

VDSers – looks like Thao, Yiling and Radhika were successful in transforming the expression plasmids of CA7, PTP1b and SCP1 in the BL21(DE3) cells! 🙂

So, everyone that is on track for the expression this week – should plan on setting up your overnight expressions  and prepping your flasks tonight for the Mentors or yourself to use tomorrow.

I’ve got a fish in the oven, if anyone’s interested.

Whenever I have to use the autoclave, I always picture this episode of Salad Fingers:

http://www.youtube.com/watch?v=cuCw5k-Lph0

 

(The autoclave reminds me of a giant oven.)

On Friday, I went through Beckman centrifugation and autoclave training and did a practice run of OD600 measurement.

Saturday 12:56 p.m: I was actually looking forward to a day in lab because I was confident about using the centrifuge and autoclave (I don’t slam the autoclave door anymore!!)

Saturday 12:59 p.m: I found my two 250 ml LB flasks shaking in the incubator, their glass bodies completely devoid of life (colony growth).

I left them there… hoping they would grow up the next day, and proceeded to virtual screening.

I came in on Sunday and found nothing but LB in my flasks, so I decided to attempt overnight growth again. Instead of autoclaving the flasks, Adam told me I could microwave them, which I did.

Now that I think about it, my transformed cells probably didn’t grow because I didn’t let the LB cool down enough before putting my cells into the flasks.

Protein Expression

A new version (Version 3) of the protocol protein expression SCALE UP has been uploaded to GDocs.

Be sure to strategize with Dr. B or a mentor before doing this step. If your class schedule does not allow you to fit this in, we can coordinate with the mentors to help you do a few of the steps.

Article

Have you guys ever wondered why all of the enzymes you are studying are cytosolic?

http://www.physorg.com/news/2010-10-breakthrough-chaperone-copper.html