Top
 
PaperToolbox
MolBio
 











 

What's new at ScienceLauncher



Photos

QuotesNew

Random Online Tools for
Molecular Biology (iGoogle Gadget)
Add to Google


Tools:

 
 
 

Most Popular Scientific e-Tools


Arrow Journal List

Arrow OD260 DNA Concentration Calculator

Arrow Oligo Calculator

Arrow DNA to Protein Translation Calc

Arrow Sample Cloning Project

Arrow Wallpapers

Arrow Periodic Table (detailed)

Arrow Protein GURU

Go to all ScienceLauncher's tools


ScienceLauncher's
Biochemistry & Molecular/Cell Biology
Blog



How many nucleotides should I leave at the 5'-end of my primer for restriction enzymes (RE) to be able to efficiently cut the amplified insert?

Visit this PDF file from NEB that has enzyme-specific information about this:

cleavage_linearized_vector_old.pdf



How to get better ELISA results?

1) Use DPBS instead of PBS for your capture antibody.
2) Filter all of your solutions.
3) Use high quality BSA from Sigma.



A useful resource:

http://snapshots.cell.com/



How to get better phospho-blots?

If you are trying to detect phosphorylation of a protein using a phospho-specific antibody and get an unsatisfactory weak signal, what usually helps is supplementing 1 uM of microcystin-LR to the lysis buffer just prior to cell lysis.



Strong western blot stripping protocol (home-made):

Strong Stripping Buffer: 1% SDS, 63mM Tris-HCl, pH 6.8, 0.5% Tween 20, 0.7% beta-mercaptoethanol (BME).
For 1L: 31.5 ml of 2M Tris-HCl stock, 100 ml of 10% SDS stock, 5 ml of Tween 20, 863.5 ml dH2O. Add BME fresh: 70 ul per 10 ml of the buffer.

1) Rinse the membranes with TBST (5 min, while shaking).
2) Incubate the membranes for 5 min at room temp in 0.2M NaOH.
3) Incubate the membranes for 30-60 min at room temp in Stripping Buffer.
4) Rinse the membranes with TBST (3 times, 5 min each, while shaking).
5) Optional: block as usual.
6) Continue with primary antibody incubations.





Here is a trick I came up with to get cleaner phospho or total blots (if your antibody gives you non-specific bands and/or high background):

1) Run 10-12 lanes of the most commonly used cell lysate (e.g. HEK293 lysate): 20-30 ug per lane.

2) Transfer onto a nitrocellulose membrane.

3) Cut out the region (a thin horizontal strip) that corresponds to the MW of your protein.
You will end up with 2 pieces of membrane (unless your protein is >250 kDa or <20kDa and it's just easier to cut off the top/bottom of the membrane).

4) Block the membranes with 5% milk/TBST for 1hr.

5) Incubate the membranes with your antibody solution at 1 ug/ml in milk or BSA O/N at 4C (it's best to supplement the solution with 0.02% NaN3 as a preservative).

6) Use this antibody solution for your actual experiment.

This negative selection will clean out most of the non-specific species present in your polyclonal antibody pool.




Why do we put EDTA into lysis buffer?

EDTA is a divalent cation chelator. There are many enzymes that would not function without their cofactors and in many cases these cofactors are divalent cations such as Mg++. Mn++, Ca++ and Zn++. If a lysis buffer is supplemented with 1-10 mM EDTA, then metalloproteases, nucleases, phosphatases, kinases and several other types of enzymes will be inactive. This is done to preserve the post-translational modification state of the proteins at the moment of cell lysis or to prevent nucleis acids from being degraded.

 


[ Homepage] [Tell Friends][Our Button - Link to us] [ Our favorite Links] [Contact Us]

[Scientific eBooks] [ e-Tools for Scientists][ Scientific Photos] [ Journal Club Pal] [ Shop] [ Multimedia] [ Quizzes]
[
GRE Subject Biochemistry, Cell and Molecular Biology eBooks and Practice Questions]
[ GRE Subject Biochemistry Blog ]
[ PCR (Polymerase Chain Reaction) Troubleshooting, Optimization, Tips and Tricks]
[
ez-PubMed ][ Index of Named Things in Chemistry and Physics]

Tools:
[Antibiotics] [ Business eBooks] [ Story eBooks] [ Cloning] [ Chemical Densities] [ Dideoxy Sequencing] [DNA to protein calc]
[
DNA Microarrays] [Health and Fitness e-Books] [ Scientific Icons] [ Western Blotting (Immunoblotting) ] [ Immunoprecipitation]
[
OD260 DNA concentration determination calculator ] [ SDS-PAGE] [ Yeast Two-Hybrid] [ RFLP] [ Plasmid Construction]

Multimedia - Flash Animations, Videos and Movies:
[
Reporter Constructs] [ Screening Oligonucleotide][Phage Display Technology]