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Fluffy's Content

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#148168 Blocking solution mistake, please help!

Posted by Fluffy on 16 January 2013 - 08:08 AM in SDS-PAGE and Western Blotting

Great! that made me feel better about my work. I guess I will find today how my results turn out. Thanks :)



#148140 Blocking solution mistake, please help!

Posted by Fluffy on 15 January 2013 - 05:56 PM in SDS-PAGE and Western Blotting

Hi all,

This is a quick question, for my blocking step in western blotting I used 5% skim milk. Instead of dissolving the skim milk in 1X TBST, I made a mistake and dissolved it in 1X TBST containing 5% BSA (which I normally use to dilute my antibodies). I went on and incubated the membrane overnight with primary antibody. I can't stop thinking about, I am worried, will my mistake affect my result? Please help!

Thanks all!



#147491 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 04 January 2013 - 09:12 PM in Protein Expression and Purification

Hello Andreea,

Oh nice hope your vacation went well. It is always good to have rest. I took a vacation too :). Thanks for getting back to me dear. I will take your ideas into consideration for the next steps of my work. I recently ran a western to confirm my protein sizes. Before I post my pictures to get you to look at them. I have a couple of questions, I want to make sure I am calculating my protein sizes in kDA correctly.

First off, as you remember I have two genes that I work on and expressed. My genes are UBC3 and UBC1.

UBC3 cDNA sequence + cloning primers
1 ctcttcttcc atttctttca aaattaaagt attgttactc tgctattggc tcaaaacctc
61 tgcaatctcc gtctccttca atttcaactc aagcaaatcc acctctttca ctagtttcat
121 cactttcaga tcagggtttg gagttgaagg tacggggggc taattgatgg cgtcgaagag
181 gatattgaag gagctcaagg atctgcagaa ggatcccccc acatcatgca gtgctggtcc
241 agtggcagag gatatgttcc attggcaagc aacaatcatg gggcctaccg atagccctta
301 tgctggaggt gtatttttgg tttcaattca tttccctcca gattatcctt ttaagcctcc
361 aaaggttgcc ttcagaacta aggttttcca tcccaacatc aacagcaatg gaagtatttg
421 tctggatatt cttaaggagc agtggagtcc agcattaacc atatccaagg tcctgctgtc
481 catctgctct ctgttgacag acccaaaccc agatgatcct cttgtacctg aaattgctca
541 catgtacaag actgacaggg ccaaatacga aaccactgct cgtagctgga ctcagaaata
601 tgcaatggga tgatgcgcaa aatgtctcca ggcatgtctg ggactttgta acagcaatgt
661 cttatgtgct tggggtgaat gaataaattc cgtgaaagaa cttagttact tcttaatctc
721 ccttcatgag ggttgttaag ggaacagctg ttttcaattt gtgaatattt atttgatgac
781 tagtaaggga gaaactgcaa tgtaattcta ctttgtttgc cagtt
Primers
Forward Primer (NdeI)
5’ c a t a t g a t g g c g t c g a a g a g g a t a t t 3’
Reverse Primer (XhoI)
5’ c t c g a g t c a t c c c a t t g c a t a t t t c t g 3’
UBC1 cDNA sequence + cloning primers
1 gcacgagggc gacttttgca taaaccaaaa ttagaatcaa attggaagag agaaaaaaaa
61 tggtggactt ggctagggtt caaaaggagc tccatgaatg caacagagat gttcaggttt
121 ctggaattaa tgttaccctt aaaggtgaca gtctcactca cttgattggt acaatccctg
181 gtcctgttgg tactccttac gaaggcggta ctttcaagat cgatatcact cttactgatg
241 gctacccatt tgagcctcca aaaatgaaat tcgccacaaa agtttggcat cccaacataa
301 gtagtcaaag tggagcaata tgcctagaca tcctgaagga ccagtggagc ccagcactaa
361 ctctcaagac agctctcctt tctatacaag cattactttc tgctcctgaa cctgatgatc
421 cacaagatgc agttgttgca cagcagtatc ttagagaaca tcagaccttt gtcggcacag
481 ctcgttactg gactgagact tttgcaaaaa catccacact tgctgcagac gacaagatac
541 aaaagcttgt ggaaatgggc tttcctgaag ctcaagtgag gagtactttg gaagcaaatg
601 gttgggatga aaacatggct cttgaaaagc tgttgtccag ctaaaaccct tctactgcaa
661 ctcatatttt gataagacaa ttatatcctt ccagcaaaag ctgatgacta gaatagagtc
721 actcggttat actgttgctt ggcaatcttg tttctgtctc ctttatggtt tgctgttgac
781 atctcttcat atcctgtgaa gattctgatg ttatttttac aatatcaagc aaattgcata
841 tgaatcatgg ggaggaagtg gactttccgg ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa
901 aaaaaaaaaa aaaaaaaaaa aaa

Primers
Forward Primer (NdeI)
5’ c a t a t g a t g g t g g a c t t g g c t a g g g t 3’
Reverse Primer (XhoI)
5’ c t c g a g t t a g c t g g a c a a c a g c t t t t c a 3’

Using google I found protein translation tools, I translated the above cDNA sequences into protein sequence. Then using google search I used a protein size calculation tool to estimate protein sizes of the above two genes. My estimated protein sizes are 16.52kDA for UBC3 and 21.38kDA for UBC1. Please do you think these are accurate not necessarily perfect? I need to know the best estimated protein sizes for the two genes for my western blot please. Also for estimating the protein sizes for western do I need to put into consideration the size of the His-tag?

Thanks Andreea so much :)



#146639 Trouble estimating my protein size using ladder (image included)

Posted by Fluffy on 12 December 2012 - 11:28 AM in SDS-PAGE and Western Blotting

Thanks for your reply John.

Ok I am waiting on my anti-his antibody and purification kit and will do as suggested. I hope for the best.
Thanks again :)



#146538 Trouble estimating my protein size using ladder (image included)

Posted by Fluffy on 10 December 2012 - 07:25 PM in SDS-PAGE and Western Blotting

Hello Everybody,

I transformed my pET14b vector containing my target insert into Bl21(DE3) cells and induced my culture with IPTG. I ran an SDS-PAGE to check for the solubility, expression, and whether my protein got induced. The different fractions cellular fractions I ran on gel include total cell protein fraction and soluble cytoplasmic fraction.

My problem is I cannot locate my protein on the SDS-PAGE gel. My protein size is approximately 21.38 kilodaltons. I cannot really know how my inductions really went? Can anyone provide me with feedback please. I would greatly appreciate it.

My image organization
far left-ladder, total cell protein induced, total cell protein uninduced, soluble cytoplasmic induced, soluble cytoplasmic uninduced-far right.
Here is the image12.10.2012 SDS UBC1.jpg

For ladder image here is the link http://tools.invitro...ned_Std_man.pdf

Thanks again!



#146248 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 04 December 2012 - 07:16 AM in Protein Expression and Purification

Thanks for your response Andreea. Hmm...when you say beads you are referring to purification step correct, then run the eluted sample on SDS-PAGE? I am planning on doing that as well but I am waiting on a kit on order. And this would most likely be a really important step and what my professor would like to see. He wants to see if possible what other proteins interact with my protein of interest after I purify my his-tagged proteins using a column and run the eluted sample on SDS-PAGE gel. According to him that if any proteins do interact with my his-tagged protein they would get pulled down (eluted sample), and should appear as well on the SDS-PAGE along with my his-tagged protein. I am looking forward to that step.

I am not sure though if I will be using the same beads as you for purification. My professor will order HisBind Purification Kit by Novagen. It is compatible with the Popculture Reagent and the BugBuster Master mix reagent that I used to extract my soluble and insoluble fractions.
I guess that should still be fine. Also another question, the past cell paste extractions that I used to run my SDS-PAGE gels are all used up. I have more cell pellets stored from the same induction experiment, it would be fine to do other round of total cell protein, soluble, insoluble protein extractions and use those extracts for my purification step? I mean the cell pellets are from the same culture I just aliquoted them in epp. tubes and harvested their cell pellets.

Sorry I ask too many questions :( this forum is my only source of support

Thanks Again :)



#146120 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 01 December 2012 - 11:28 AM in Protein Expression and Purification

Hello I am back

I did an SDS-PAGE that included total cell protein fraction, cytoplasmic soluble fraction, and cytoplasmic insoluble to test the expression.
My ladder is suppose to have a pink orientation band at 64kDA. The link to it is http://products.invi...roduct/10748010 But for some reason the pink band did not appear on my ladder so it is kind of hard for me to figure out my protein band. My protein size is ~16.52kDA.

The organization of my SDS-PAGE image below is
far left-ladder, uninduced total cell protein, induced total cell protein, uninduced cytoplasmic soluble, induced cytoplasmic soluble, uninduced cytoplasmic insoluble, induced cytoplasmic insoluble-far right.


11.30.2012 UBC3 100uL readjusted.JPG

It is kind of hard for me to distinguish my protein band..any suggestions as to which is mine? Also if it is there is my expression good, can I go on?

As you can see also the insoluble fraction shows nothing at all?? Is that normal?

Please provide me with some feedback.
Thanks All Posted Image



#145492 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 18 November 2012 - 08:24 PM in Protein Expression and Purification

Andreea I am not sure about the second part. I am sort of confused as to how you do it. But can I follow the pET manual protocols even if I centrifuged down my culture and stored as a pellet?
I guess I can since I centrifuged down 1ml culture aliquots into pellets and stored at -80C. In the pET manual it says to take 1ml aliquots and work with those. I am just worried because I stored my pellet and it does not mention to store the pellet in their protocols.



#145392 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 16 November 2012 - 05:09 PM in Protein Expression and Purification

Sorry in addition to the question I posted earlier about OD600 readings, I also have another question about the future steps. I remember in one of the posts Andreea mentioned that after a successful induction I can spin down (centrifuge) my culture to obtain pellets that can be stored at -80C until analysis. For analysis I wanted to compare the total cell protein fraction in parallel with other fractions such as the medium fraction, soluble cytoplasmic fraction, and the insoluble cytoplasmic fraction for the recovery of my target gene. In the pET manual 11th edition, there are protocols on how to isolate those fractions and then analyze them by SDS-PAGE. I wanted to follow their protocols since they are well written and explained but the problem is I won't have time to do it on the same day. I would have to as Andreea mentioned spin down the cells and store the pellet at -80C. If that ends up to be my case, can I still use my stored pellet to follow their protocols in the manual. I am worried since in their manuals, the protocols for the extraction of the fractions mentioned above always have as their first step: Prior to harvest take a 1ml aliquot of the culture..etc..
And this is not my case since I harvested the cells (pellet at -80C). What do you think I would have to do?
I hope I was clear enough I am sorry if this is confusing.
Thanks Posted Image



#145363 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 16 November 2012 - 06:48 AM in Protein Expression and Purification

I am still confused about OD600 measurement, and I am worried that I did it wrong last night.

I will clearly indicate my steps. I am using a cuvette spec.

For blank, I took 1ml fresh medium and diluted by adding 3ml water
For bacterial culture samples, I took 1ml of bacterial culture and diluted by adding 3ml water as well.

The cuvettes I am using are ones that light beam can pass through all sides. I made sure it is clean.
I loaded 1ml of my diluted samples in the cuvettes.

I blanked the machine and Abs. was zero.
I measured my diluted samples which I got negative Abs. for them Posted Image

Looking at the cuvettes it seemed like the 1ml was not enough and I thought to myself that light is possibly not going through the solution at all to give a reading.

I ended up doing it again by filling cuvettes with 3ml of the diluted blank and the diluted bacterial culture samples (prepared above).
For blank I got zero.
For samples since I had two differet samples, one I got Abs. 0.321 which I multiplied by 3 (dilution factor)= 0.963, and the second one I got an Abs. of 0.360 which I also multiplied by 3 (dilution factor)= 1.08

Based on these Abs. I moved on to the induction step.

But I was thinking about it the whole night and I have a feeling that I am not correctly measuring OD600.

Please help me out, I am stressed about this.

Thanks all Posted Image



#145218 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 14 November 2012 - 07:47 AM in Protein Expression and Purification

Thanks John :) That actually made me feel better about my transformation. Okay I will do just like you then, I will inoculate two colonies. Thanks for the hints.

All the best to you :)



#145211 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 14 November 2012 - 06:55 AM in Protein Expression and Purification

Hello again,

So I did my step 1 of my protocol yesterday (transformation and plating). The plates have different plating volumes (100uL, 200uL, 300uL, and 400uL). I am not sure if my transformation worked the way it should. I mean I have ALOT of small colonies on my plates. So do you think I should move on to step 2 today? And if so, do I choose any single colony from any plate and inoculate that? Please respond back to me on this.

Thanks again :):)



#145123 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 12 November 2012 - 05:43 PM in Protein Expression and Purification

Ok hmm I can't really access the manual..hehe..do I have to buy it inorder for me to see its contents? Is there another way possibly.



#145119 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 12 November 2012 - 05:06 PM in Protein Expression and Purification

Perfect idea Andreea. Thanks :)

For analysis, I will be doing SDS-PAGE with coomassie blue staining first, then Western Blotting. I will focus on SDS-PAGE now. I noticed different protocols online. I do not know which is best. I think it also depends on the protein size. My protein sizes are 16.52KDA and 21.38KDA. Now, what do you normally prepare in terms of the sample loading buffer concentration, running buffer, stacking gel ,and running gel? Oh and do you normally prepare ahead of time and store at a certain temperature, and use them when needed.

Sorry all this is new to me, so please bear with me.



#145118 Tomato Glucanase and Glutamine Synthetase Assays

Posted by Fluffy on 12 November 2012 - 04:51 PM in Biochemistry

Thanks for answering my questions. I will put an empty plate in the reader and compare the readings and hope for the best.

Cheers :)



#145022 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 10 November 2012 - 08:02 PM in Protein Expression and Purification

Hi again,

A question came to my mind and thought I should ask about it to know the answer ahead of time :)

After step 6. of my protocol, the culture I get (that is the one I incubated with vigorous shaking at 18C for 16-20hr) can it be stored for a couple of days? I want to use it to get total cell protein fraction, the soluble and insoluble cytoplasmic forms and run on SDS-gel, but I do not want to do it the next day after the 16-20hr incubation. Is there a way to store that culture for a couple of days and continue my work later with it?

Thanks everyone :)



#144988 Tomato Glucanase and Glutamine Synthetase Assays

Posted by Fluffy on 10 November 2012 - 07:31 AM in Biochemistry

Sorry Bob in my earlier post I said their absorbance was subtracted from that of the blank, I meant to say the blank's absorbance was subtracted from their absorbance.

About the proteases, I use PMSF in my extraction buffer which should inhibit proteases. Could it be dirt like my microplate being dirty which intereferes with the readings? I am so confused

Also, I don't know if this affects but for my protein samples, I have taken the supernatant after the centrifugation step, although I can still see some residue (really tiny green stuff-leaves maybe) in there.

Freezing/thawing my protein can have an affect you think?

Thanks again and enjoy your day!



#144955 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 09 November 2012 - 04:44 PM in Protein Expression and Purification

Oh great thanks for pointing that out! I did not know about that :). I am glad that it is a good protocol, I cross my fingers that it would actually work in the lab. I will let you know how things turn out.
You have been great help, thanks alot Andreea! Best of luck to you in everything :)
Cheers :)



#144954 Tomato Glucanase and Glutamine Synthetase Assays

Posted by Fluffy on 09 November 2012 - 04:38 PM in Biochemistry

Hi Bob,

Thanks for your reply. Hmm..for example for my glucanase assay I did have three controls, each had all the components of the assay except for the substrate, Laminarin. Surprisingly, all after substracting their absorbance from that of blank (in my case my blank is my protein extraction buffer mentioned above in my comment, Tris-HCl buffer) had negative absorbancies.

Also I am still confused why would my lysis buffer result in my negative absorbance values? I am guessing by lysis you mean the protein extraction buffer.

Thanks again :)



#144880 Tomato Glucanase and Glutamine Synthetase Assays

Posted by Fluffy on 08 November 2012 - 06:25 PM in Biochemistry

Hi Bob1,

Thanks for your reply.

I am sorry my post was not clear enough.
My tomato leaves were vacuum infilitrated with MG115 (a proteasome inhibitor), water, and FB1 (a fungal toxin). Our lab focuses on disease resistance and plant defense responses. The treatments serve as stress. We wanted to test the relative activity of certain enzymes under those stresses. The two enzymes are B-1,3-glucanase and glutamine synthetase.

I first extracted total protein by adding 1ml of 200mM Tris-HCl buffer (0.25mM EDTA, 5mM DTT, and 1mM PMSF) pH 8.0 to 0.5g of frozen tomato leaves and grinded them using a mortar and pestle. I made sure the mortar and pestle were cooled down with liquid nitrogen. The homogenate was centrifuged at 12000g at 4C for 20min.
The concentration of my protein samples was determined using the standard Bradford method.

B-1,3-glucanase
The reaction mixture consisted of 0.4ml of citric-acid phosphate buffer (pH5.6) containing 1mg/ml laminarin and 0.1ml of enzyme solution (this is my total protein samples, keep in mind that after determining their concentrations, I diluted all to the same final concentration). After 15 min incubation at 37C, 0.5ml of the alkaline copper reagent was added and the mixture was heated at 100C for 10 min. After cooling on ice, 0.5ml of the arsenomolybdate reagent was added, followed by 3.0ml of water after the development of the blue color. The absorbance was measured at 660nm.
Then to measure the relative activity, I set the water treatment as (100%) and I measured the relative activity of the other treatments with respect to water.
Ex. if water-treatment absorbance is 0.56 and the FB1 treatment is 0.97, I divided 0.97/0.56*100= 173.21%

Glutamine Synthetase (GS)
We adapted a method for wheat, even though there are publications with tomato GS methods.
GS activity was measured using the
synthetase assay based on the method described by Lea
et al. (1990) and optimized for wheat leaves as follows.
100uL of crude leaf extract (the same protein samples were used as the ones used in glucanase assay above)was added to 380uL of assay
mix which consisted of 100 mM TEA, 80 mM glutamate,
6 mM hydroxylamine HCl, 20 mM MgSO4, 4 mM EDTA
at pH 7.6. The reaction was started by the addition of 20 uLof 0.2 M ATP at pH 7.6. After 10 min of incubation at
30C, the reaction was stopped by the addition of 500 uL of
ferric chloride reagent (0.24 M TCA, 0.1 M ferric chloride,
1.0 M HCl). Samples were then centrifuged at
10,000 g for 5 min and absorbance read at 505 nm.

We also looked at the relative activity of the enzyme for the different treaments and it was done the same way as that in the glucanase assay above.


I am sorry Bob as this is really long but I wanted to make sure that I am very clear on this. Please if you have more questions or not sure about anything, let me know :)

Thanks again :)



#144844 Tomato Glucanase and Glutamine Synthetase Assays

Posted by Fluffy on 08 November 2012 - 09:46 AM in Biochemistry

Hello Everybody,

Can anyone help me understand why I am getting negative absorbance values in both assays for some of my protein samples?

By the way I am looking at the relative activity of the enzymes, I am not using a standard to measure their activity.

Thanks for all Posted Image



#144840 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 08 November 2012 - 08:49 AM in Protein Expression and Purification

Hello again :)..

Awesome Andreea..I appreciate your time with this:)
I sat down and made a protocol of what I will do next week willingly. If anyone thinks wants to add or make changes to my protocol please let me know, all your comments would help.

1. Plate my transformations on LB-Agar plates containing Carbenicillin and Chloramphenicol antibiotics.
2. Inoculate a single colony with 3mL of LB media containing Carbenicillin and Chloramphenicol (it will also have 0.5-1% glucose to reduce basal expression levels, pH will be around 8.2 to overcome low pH problems) in a culture tube.
3. Incubate the culture prepared in step 2 at 37C at 250rpm overnight.
4. Next day, add the 3ml culture to a 100ml of LB media containing carbenicillin antibiotic only.
5. Shake the culture prepared at step 4 at 37C for 2-3hours until OD600 is 0.5-1.0. Then cool the culture to 18C.
6. Before induction, divide the culture from step 5 into two 50ml cultures. One of them will serve as the uninduced control. The other 50ml will be induced by adding IPTG to a final concentration of 0.4mM. Both cultures will then be incubated with vigorous shaking at 18C for 16-20hr.

I will also keep in mind to use proper culture tubes and flasks and try to keep volume 10%-20% of tube or flask volume for better aeration.

What do you think :)

Thanks and have a nice day!



#144743 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 06 November 2012 - 06:11 PM in Protein Expression and Purification

Thanks for your replies.

I am sorry Andreea for my misunderstanding :)
I want you all to be patient with me. I am hesitant to start this. Since I am a fresh starter, I will need your feedback on this.
I read websites and papers (including the papers you sent me Andreea-they were really helpful :)) and they all have slightly different ways of starting their work.
I understand that I have to first transform my pET14b vector in BL21-(DE3)RIPL cells (Andreea when plating I will put both antibiotics correct?), then I will take a single or two colonies and inoculate with LB medium (Andreea this will have both antibiotics as well correct?) at 37C overnight. Now I am not sure what is the best volume of LB medium to use at this point? I saw different volumes being used by different websites and papers..After that I add that culture to LB medium (Andreea this time I only put Amp antibiotic correct?)..but samething here I am not sure for a starter like me what would be the best volume of LB medium to add the culture to?...
Note that I will be dividing it into an induced and uninduced control

Next would be growing it to midlog phase (OD600 0.5-1.0)-what is the best way to measure it? I have a microplate reader and is it best to measure it at different time intervals throughout the 2-3hours of growth..and should I dilute my aliquots to get accurate readings?..

For now these are my concerns..Sorry for my really long inquiries ..

Thanks all :):)



#144704 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 06 November 2012 - 07:33 AM in Protein Expression and Purification

John: Thanks for the reply. I will use Carbenicillin instead of Ampicillin. Thanks for providing the concentrations you have tried in your lab.

Ascacioc: hmm..just to be clear that I understand what you said..you prefer that I only use Ampicillin (or carbenicillin) for both my LB medium and LB-Agar plates during my protein expression work. You don't think it is a good idea to use both antibiotics as in Amp and Chloramphenicol?
Correct?
And thanks to you dear :)



#144657 pET14b vector with insert transformed in BL21(DE3) RIPL cells

Posted by Fluffy on 05 November 2012 - 07:00 AM in Protein Expression and Purification

Hello Everyone,

Hope you are all doing fine.

I will be starting protein expression work soon. I am currently setting up an experimental design to get started. I came across an obstacle and would love to hear your inputs about it.
I will be first transforming my pET14b plasmid (has my target insert) in BL21(DE3) RIPL cells. Now, my vector has an Ampicillin resistance marker. The cells also have their own vector that possesses an antibiotic resistance marker called Chloramphenicol. When plating should my LB-agar plates contain Ampicillin or Chloramphenicol? I personally thought it should be Ampicillin (or carbenicillin-can be used instead of Amp) so that way only the cells with my pET vector survive.

Is that correct?

Thank you :)




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