Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!

Pangea's Content

There have been 97 items by Pangea (Search limited from 20-February 19)



Sort by                Order  

#163303 Bradford Assay Calculation! Help!

Posted by Pangea on 11 December 2013 - 12:21 AM in General Lab Techniques

Missle thank alot can you explain me again why ı should not divide by the volume? I also remember that is not necessary. Can you give me an example? How much of the unkown sample in volume must be pipeted into the 1ml Cuvet?




#161940 Bradford Assay Calculation! Help!

Posted by Pangea on 31 October 2013 - 11:44 AM in General Lab Techniques

Dear All,

 

I have simple problem about Bradford Assay Calculation. I not designed this and would never do it in this way.

 

(BioRad) BSA Standard Curve

 

Stock Solution: 1.48 mg/ml

 

1: 5.92 ug/ml (from Stock 4 ul into 796 ul H2O + 200 ul Dye Reagenz )   OD595 : 0.434

2. 8.88 ug/ml (from Stock 6 ul into 794 ul H2O + 200 ul Dye Reagenz)    OD595 : 0.657

3: 11.84 ug/ml (from Stock 8 ul into 792 ul H2O + 200 ul Dye Reagenz)  OD595 : 0865

4: 14.8 ug/ml (from Stock 10 ulinto 790 ul H2O + 200 ul Dye Reagenz)   OD595 : 0.989

 

y = 0.23x + 0.177

R2 = 0.99

 

Lets take 6 ul an unkown sample without dilution and a absorbence of OD595 0.295:

 

0.295 = 0.23 x +0.177 ----> (0.295-0.177)/0.23 = x ---> 0.513 mg/ml ATTENTION: We dived by 6 ul to obtain the true conc. : 0.513/6 = 0.085 mg/ml.

 

Can you confirm the calculation neglecting to way it was generated.?

And we do not have a sample 0 the blank is not in the graph.!

Must we dived by 6?

Can you send me a protocol?

 

 

 

 

 

 

 

 

 




#154198 Bacterial Media Formulation

Posted by Pangea on 24 April 2013 - 10:33 PM in Protein Expression and Purification

BL21 DE3 or Origami ? But if you pass me all formulation what you it would be great. I think i will try auto-induction media with 2 mM MgSO4 or higher. Some Trace Elements. I know LB, TB, SB, M9 etc. but some how people have good media formulation. Actually, I get with 2xTB at 37 C and 0.5mM İPTG an OD600 of 14 in a shaking flask (100 ml/500ml). Any suggestions?



#153915 Bacterial Media Formulation

Posted by Pangea on 18 April 2013 - 12:09 AM in Protein Expression and Purification

Dear All,

I would like to reach a OD600 greater than 50 in flask and 100 in Fermenter. Do someone knows a good media beside LB, TB or SB?

Thanks in advance

All the Best



#152180 Production of GST tagged fusion proteins in BL21 Star cells

Posted by Pangea on 13 March 2013 - 12:18 PM in Molecular Biology

One question to you construct: Do you have additional amino acids after cleavage of your protein from your gst?



#149918 Interferon Alpha

Posted by Pangea on 08 February 2013 - 10:34 AM in Protein Expression and Purification

Hi Everyone,
I can not express interferon alpha in bl21 cells. Does anyone have an idea or protocol. Vector is pET9a.
THANKS in Advance



#148128 Manual/ User guide for opticell from thermoscientific

Posted by Pangea on 15 January 2013 - 12:16 PM in Tissue and Cell Culture

https://www.google.d...lient=firefox-a



#148092 western blot troubleshooting

Posted by Pangea on 15 January 2013 - 08:26 AM in Molecular Biology

Welcome.

as almost says, we need more detail and specification of your problem. But it seems that you just have different amount of actin as a internal control. Thats because your loading conc. are different when you perform SDS-PAGE.You can calculate the amount maybe with densitometric methods.



#148091 protein sample floating out of the tricine gel

Posted by Pangea on 15 January 2013 - 08:19 AM in Protein and Proteomics

Check Loading Buffer again! Bromophenol Blue!



#148006 Alternative to pET Vectorsystem

Posted by Pangea on 14 January 2013 - 08:28 AM in Molecular Cloning

There is no big problem with pET. Just in case.



#147980 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 01:35 PM in Molecular Cloning

I guess you use Calcium Chlorid and using all tranformed E. coli to plated on Amp plates.



#147977 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 01:28 PM in Molecular Cloning

Better to increase insert. On your Gel you dont see your expected size? Maybe difficult on this size of an insert. With recutting i mean where do you no that its not working?



#147974 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 12:41 PM in Molecular Cloning

I already changes my mistake. Sorry.



#147972 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 12:33 PM in Molecular Cloning

XbaI is dam sensitive. Spe1 is compatible with Xba1. Maybe, use klenow and ligate blunt end. Did you do Re-cutting? PCR with Taq or Pfu? Generally, try different ratio for ligation.



#147963 Stem Cell Donation

Posted by Pangea on 13 January 2013 - 09:40 AM in Stem Cell

Cheers on both.



#147962 pET 32 (+)

Posted by Pangea on 13 January 2013 - 09:38 AM in Molecular Cloning

What means the "+" in the vector description? What kind of orientation is it?



#147961 Alternative to pET Vectorsystem

Posted by Pangea on 13 January 2013 - 09:35 AM in Molecular Cloning

Any suggestion about other vectorsystem than pET?



#147958 High Yield Protein Expression 10g/L

Posted by Pangea on 13 January 2013 - 09:30 AM in Protein Expression and Purification

So you mean replacement by centrifuging the cells and renewing the media. Can you give me more details like an protocol?



#147957 Stem Cell Donation

Posted by Pangea on 13 January 2013 - 09:28 AM in Stem Cell

Germany



#147950 isolating DNA from cells in suspension

Posted by Pangea on 13 January 2013 - 07:51 AM in Molecular Biology

Getting rid of unnecessary molecules in media. And handling with less solution (concentrating).



#147949 Software for scanning of specific aa sequence in proteins

Posted by Pangea on 13 January 2013 - 07:45 AM in Bioinformatics and Biostatistics

Check this:

http://blast.ncbi.nlm.nih.gov/



#147948 Stem Cell Donation

Posted by Pangea on 13 January 2013 - 07:40 AM in Stem Cell

Where can I type me and donate stem cells?



#147947 Purifying a "protein"

Posted by Pangea on 13 January 2013 - 07:12 AM in Biochemistry

Run a SDS-PAGE and do Silver-Staining. Measurement on 280 nm for Purity.



#147946 Suggestion for a book!

Posted by Pangea on 13 January 2013 - 06:49 AM in Immunology

Listen type into google search as follows: Cytokine filetype:pdf

The part filetype will give you just PDF files and gives you a smile in your face:). You can do this also with doc, xls, etc. Enjoy the discovery.


Books:
Cytokine Protocols (Methods in Molecular Biology)

http://elib.fk.uwks....ogy Series].pdf

http://www.ctf.edu.t...1_Cytokines.pdf



#147945 How to raise polyclonal antibodies against whole bacteria live &/ or inactiv

Posted by Pangea on 13 January 2013 - 06:41 AM in Immunology

Thats just general information, but which bacteria you want to produce rabbit Ab. It will be take 3 month i guess in rabbit. Whole bacteria means just its capsule, isnt? You might look up the consistence of the capsule. It dosent matter if it Gram-negative or -positive, isnt? But i guess it will be unspecific Antibody. Do your Bacteria has specific marker molecules on his surface?

http://www.uniprot.org/keywords/875

http://ecvam.jrc.ec....hopReport35.pdf




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.