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## Pangea's Content

There have been 97 items by Pangea (Search limited from 10-April 20)

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Posted by 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?

Posted by 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.

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?

### #154198Bacterial Media Formulation

Posted by 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?

### #153915Bacterial Media Formulation

Posted by 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?

All the Best

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

Posted by 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?

### #149918Interferon Alpha

Posted by 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.

### #148128Manual/ User guide for opticell from thermoscientific

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

### #148092western blot troubleshooting

Posted by 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.

### #148091protein sample floating out of the tricine gel

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

### #148006Alternative to pET Vectorsystem

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

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

### #147980Difficult Ligation - Details Inside

Posted by 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.

### #147977Difficult Ligation - Details Inside

Posted by 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?

### #147974Difficult Ligation - Details Inside

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

I already changes my mistake. Sorry.

### #147972Difficult Ligation - Details Inside

Posted by 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.

### #147963Stem Cell Donation

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

Cheers on both.

### #147962pET 32 (+)

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

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

### #147961Alternative to pET Vectorsystem

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

Any suggestion about other vectorsystem than pET?

### #147958High Yield Protein Expression 10g/L

Posted by 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?

### #147957Stem Cell Donation

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

Germany

### #147950isolating DNA from cells in suspension

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

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

### #147949Software for scanning of specific aa sequence in proteins

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

Check this:

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

### #147948Stem Cell Donation

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

Where can I type me and donate stem cells?

### #147947Purifying a "protein"

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

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

### #147946Suggestion for a book!

Posted by 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

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

Posted by 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