Fluffy

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

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

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

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 His•Bind 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

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   23.42K   41 downloads

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