Western trasfer and blotting troubleshooting

shifali chatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Sun May 3 22:47:07 EST 2009


Dear All!
I transfered the protein on 12% Tris -Tricine gels to Methanol wetted PVDF membrane using semi-dry apparatus at 300A, 9V for 40 min.
Transfer buffer: 48mM Tris,Glycine 39mM pH(8.9) without adjusting,+ 20% methanol.
After transfer, markers on one side(first lane) got beautifully tranfered but the other side of the gel (last lane),got diffused, can anybody suggest why?Anyhow, I proceeded for blotting.

I dot blotted few samples on the membrane and allowed to air dry, because, a day before, these dot blots worked for these samples.I rewetted the membrane with methanol and washed with water and blocked.

After Blocking in non-fat milk, I used 1:2000 anti-His mouse monoclonal, followed by 3X washes in 20mMPB +254mM Saline (PBS)+ 0.1% Tween 20 and 3X washes in PBS. Then I added 2 Ab,anti-mouse preadsorbed human, 1:5000 in Blocking buffer, it got pptd. on ice and i notice only after adding on blot. I kept the blot at 37/shaking forsing 15-20 min. and ppt. got dissolved. After 1 h incubation, I again washed as above.
I noticed that two proteins from prestained marker lane are missing.  Then, developed using DAB, ( Tried ECL on diffused side of the gel, last 3 lanes). I could not find any signal with both the techniques.
I have standardized the antibody dilution before proceeding for this. It works.Whatever, when different I mentioned, the pptn.happened and I kept at 37. ealier, i was using 0.01% Tween 20. this time, i used 0.1% Tween 20? Then what could have gone wrong?
It is the 2Ab ?
Is it the pptn.?
Why I see diffusion during transfer?

Earlier, i had used, goat anti-mouse HRP, this time it was preadsorbed.

Can anybody suggest where is the room for error that failed my expt.?

Thanks
REgards
Shifali

On Wed, 29 Apr 2009 17:55:49 +0530  wrote
>Shifali,
>
>the N-end rule works at the cytoplasm; it has no influence on the
>half-life of proteins secreted into the ER (as should be the case for
>your venom protein in Pichia). Also (at least in E. coli) remember that
>the amino-terminal methionine is not always removed by MAP; this depends
>on the bulkiness of the side chain for the second a.a. Unsurprisingly,
>residues which are destabilizing according to the N-end rule often
>inhibit removal of the N-terminal Met by MAP when they are in the second
>position.
>
>how did you check for expression in yeast? did you also test cell
>lysates? Straight SDS-PAGE or Western blotting?
>
>> -----Original Message-----
>> From: methods-bounces from oat.bio.indiana.edu 
>> [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of 
>> shifali chatrath
>> Sent: Tuesday, April 21, 2009 10:46 PM
>> To: methods from magpie.bio.indiana.edu
>> Subject: Expression and -N end rule
>> 
>> Dear all!
>> I had mentioned my problem earlier also in the forum but 
>> nobody replied. I am sure you great minds should have a 
>> solution to my problem. Please help me if you can!
>> 
>> I want to express a snake venom protein,~ 10kDa, 10 cys 
>> residues and 5 disulphide bonds.Mature protein has Arg as 
>> first residue after signal sequnece is clipped off.
>> First of all, i tried yeast expression system.
>> I expressed my protein in pPICZ alpha, under alpha factor 
>> signal sequence followed by Kex2 and Ste13 signal sequences, 
>> such that the mature protein will have native -N terminus 
>> with Arg as first residue, as of my protein. BUt, I could not 
>> see any protein expression even under different medium and 
>> proper aeration and whatever possible troubleshooting one could do.
>> Recently, i read -N end rule which states that Arg is 
>> destabilizing residues and directs the protein for 
>> degradation to 26S proteasome.
>> I checked with the company, they say, It hold true for every 
>> expression system.
>> But, my question is, if that is the case, how the protein is 
>> stable in snake's venom. I have found the protein in the 
>> crude venom of the snake.
>> Also, the protein is expressed under alpha factor signal 
>> sequence which is supposed to be secreted into the medium. 
>> Therefore,once the protein is out into medium, it is out of 
>> cell now, Kex2 and Ste13 cleavages should be happening 
>> outside the cell, therefore, there should be no proteasomal 
>> degradation. Am I right?
>> 
>> After clipping off signal sequence, protein will be exported 
>> out, still will there be any proteasomal targetting of 
>> proteins with destabilizing residue at -N terminus?
>> 
>> I read in -N end rule that, protein's life depends upon 
>> penultimate residue to Met. We know, for a protein to be 
>> synthesised, we need to have  a start codon,i.e. Met. In -N 
>> end rule they say, Met will be removed be 
>> Metaminopeptidase(MAP), so if we express a protein we should 
>> not be counting Met residue in the expected MW? Because, not 
>> all mature protein sequences start from Met.
>> 
>> 
>> Then,I tried expressing my protein using E. coli epression 
>> system, cDNA cloned in pET19b in Rosetta gami cells and RIL 
>> cells.I couldn't see any expression. Cells were growing 
>> normaly.(I hope, the protein is not inhibiting transcription 
>> and translation). Met and Gly had to be added at -N terminus 
>> to maintain the reading frame.
>> Protein is 10Kda and was cloned without tag sequence. I mean 
>> expected protein should be 10kDa.I confirmed the sequences 
>> before cloning and even plasmid isolated from ex-pression 
>> hosts. DNA is there but no protein.
>> 
>> Please answer any, if not all, of the above question. i am 
>> struggling with all this for alst 8 months. Your suggestions 
>> and help will be highly appreciable.
>> 
>> Thanks in advance.
>> 
>> Shifali
>> 
>> 
>> 
>> Shifali Chatrath
>> Graduate Student
>> Protein science Lab
>> Dept. of Biological sciences
>> National University of Singapore
>> Singapore
>> +65-65161210
>> +65-96393449
>> _______________________________________________
>> Methods mailing list
>> Methods from net.bio.net
>> http://www.bio.net/biomail/listinfo/methods
>> 
>

Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449


More information about the Methods mailing list