Hello,
I've been having trouble with biotynilated protein purification using Avidin-Agarose resin. The idea is to use the purified biotynilated molecules as samples for MALDI-TOF-MS.
To check the purity of the eluate, I did silver staining and Western blot. Unexpectedly, the background of silver staining is very high and I cant distinguish the discrete bands corresponding to my proteins, which appear in WB.
Also, I made a negative biotynilation control in which I have no WB signal. However, in silver staining I have no differences between biotynilated and unbiotynilated treatments (and high background...).
A summary of the protocol:
-Lyse cells with RIPA buffer
-Incubate supernatant ON with the resin
-Wash 5 times with 4V RIPA
-Elute in Cracking Buffer and 1' boiling. Take supernatant.
Any suggestions?
It's actually a pretty nice silver staining. Unfortunately, it shows no difference between the 2 samples. One possibility is that the biotinylation was unsuccessful, and all you are seeing on the gel is background non-specific binding to the resin. There may have been a very minor amount of biotinylation, which is why you can see the difference on the Western blot, but not enough to see a difference on the silver-stained gel. My recommendation would be to revisit the biotinylation procedure.
Here are some useful tips:
http://www.rockland-inc.com/streptavidin-biotin-tips.aspx
I must declare that avidin is a multiple binding protein to lipoic acid/biotin/amino acid.
Please, see files; D-asp avidin, Lipoic acid avidin, and Biotin determination.
Affinity is strongest to D,L-lipoic acid, and to D-biotin, dethio-biotin, and weaker to amino acids and L-Asp, D-Asp.
Thank you Dr Shapiro and Hayakawa for your answers.
Actually, I've titrated the biotynilation via FACS and checked the biotin levels per cell.
Probably it's neccesary to preincubate samples with unconjugated agarose beads and add a blocking step with BSA. It would be a good idea?
That does sound like a good idea, especially the preincubaton with unconjugated beads. If you preincubate with BSA, you will probably end up with a lot of BSA in the final sample, even if you wash thoroughly.
Hello again,
I´ve tried pre-incubation with unconjugated agarose and even though background is reduced, the situation remains similar.
I think maybe a blocking step its needed. Quoting Dr Hayakawa, if streptavidin binds aspartic acid (present in surface of most proteins) with intermediate affinity, blocking binding sites with biotin or decreasing resin volume should favor high affinity biotin-streptavidin interactions.
I am sorry for not yet tested of streptavidin (since streptavidin is a very high cost drug). Although streptavidin has no glycochains, but I think that the multiple binding-characteristics is essntially similar to avidin. Rat serum thiol-type biotinidase/lipoamidase kinetics (biotinyl-6-aminoquinoline (BAQ) as substrate) is allosterically effected by 0.2 mM lipoic acid, but bacterial (Lactobacillus casei (Shirota)) membrane-biotinidase/lipoamidase (BAQ as substrate) is competitively inhibited by lipoic acid (please see file; J Chrom B rat ,,,)). Although active center is different between rat and bacterium, the charasteristics of multiple hydrolase (both biotin-amide and lipoic acid-amide substrates are hydrolyzed) is similar.
I previously have used an affinity chromatography (please see the file LIP affi), and I have recognized that the affinity chromatography works less than RP-HPLC.
Then, if the protein molecular weight is less than 80,000, I would like to recommend the use of direct purification by RP-HPLC (see file RP-HPLC lysozyme). I think that proteins fractionated from the RP-HPLC column (containing phosphate ions and ,,,) may be directly applicable to MALDI-TOF-MS analysis (to use acetonitlile (AN) is only to prevent the machine from machine to be tained), however analyzing of the protein after dialysis may be more suited to maintain the machine in the clean state.
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