Hi,
it is no problem to reduce and alkylate your proteins with DTT and IAA before loading the SDS gel. There is no need for a reducing laemmli buffer afterwards but it does no harm. For example, you can use a reducing lysis buffer with DTT for your cells/tissue and alkylate with IAA afterwards. To get rid of the IAA it is best to perform a protein precipitation. When you perform in-gel digestion protocol you don't have to reduce and alkylate again.
Yes, it's fine. But isn't TCEP a reducing agent rather than an alkylating agent?
Maybe you want to use TCEP and IAA after? Having said that, if you want to use TCEP at all, you should probably use it throughout, since, as I understand it, the benefit over DTT is less side-reactions. Does that make sense?
Should be ok. If you reduce all disulfide bonds in the first step, second one will do nothing.
sure, you can reduce your proteins with DTT and then alkylate them with IAA, or yoy can use TCEP instead of DTT. I do not understand what you refer with "twice": you can reduce you protein with DTT OR TCEP before SDS gel loading and doing the protein digestion.
In my opinion, alkylation is only possible with IAA. If your first treatment is extensive, all thiols will be alkylated. Thus, the second one (DTT+TCEP) is possible, but you will reduced back even those alkylated disulfide goups, so that it could be good idea for MS as the MW of the sample and fragments are not changed by the -CH2-CONH2 groups. According to TCEP instructions, even very stable alkyl disulfides are completely reduced at room temperature and pH 5 in less than 5 min.
I am sorry for the confusion i created by typo.I did my reduction and alkylation with DTT and IAA respectively before loading gel.However to ensure that the proteins are properly reduced and alkylated i repeated the reduction with TCEP and alkylation with IAA during gel digestion.I want to make sure that by doing so i am not hurting my samples.
I prefer using TCEP for reduction of my samples (because it is superior to DTT) before loading the gel but the TCEP we buy is TCEP-Hcl from Thermo.When i make a stock of TECP-Hcl and add it to my 2X SDS sample buffer it turns highly yellow and acidic.
I am constant mystified why people feel the need to add reducing agents during electrophoresis. Those things are charged and will move in the electric field and strange reactions can be forced to happen in electric fields because reactions can be driven away from equilibrium.
Reduction and alkylation should be carried out in solution, preferably with some kind of denaturant to open the proteins up and make disulfides accessable. The reaction will be far more efficient in solution than trying to get chemicals to diffuse into a gel. We use 5mM tributylphosphine (TCEP is the same) and 10-20mM acrylamide monomers together as they don't react with each other. DTT reacts with alkylating reagents and that's why there's always two steps and a massive excess. The pH needs to be above 8. At these concentrations, the sample can go straight on a 1D SDS-PAGE without desalting. For IEF, clean-up with a BioSpin equilibrated in 7M Urea, 2M thiourea, 1% C7BzO (or 4% CHAPS if you insist on living in the past). Nothing else is required and no post gel run reduction or alkylation is necessary.
This is a different point, and in essence I would agree. But most of the TCEP action would be finished in the first few minutes, before the electrophoresis will be initiated. Anyway, your comment should be taken into account. It is a Sudhakar decission.
#Matthew, just a question, in your way ..... what 10-20 mN acrylamide monomers for? Cheers
@Matthew:Your protocol sounds interesting.My protein lysates are made using RIPA lysis buffer.I want to fractionate proteins in the lysates using Bio-Rad mini gradient gels.Maximum loading volume on these gels is 50ul.I want to load around100ug of lysate in each lane.Due to volume limitation i have to TCA/Acetone precipitate the lysates,resolubilize in 2X SDS buffer with DTT, boil at 60 degrees for 15 minutes, then cool it down to room temperature and add 5mM IAA and incubate at RT in dark for 30 mins.Then i load my sample on to the gel.
Can i apply your protocol for my purpose? And if so what is the best way to do so?
Suda\hakar - I am still unclear why you wish to repeat the reduction /alkylation step twice (to ensure the proteins are properly reduced??). The DTT/IAA steps should be adequate. To then repeat the DTT step is not sensible unless you have altered the conformation or conditions (pH etc) of your proteins in solution. Have you experienced a failure of IAA to alkylate in the past?
If so then don't use IAA at all!
The acrylamide is the alkylating reagent, better known as propionamide in UniMod. You often get acrylamide alkylation using homemade gels that have not polymerised for long enough.
Sudhakar, your protocol is fine and will work well. We often load 100-300ug of protein on Midi (Criterion) sized gels without issue, although resolution depends on the sample's dynamic range of protein concentration which you can only determine by running it on a gel. You may want to add some more IAA to at least the same concentration as the DTT as some of the IAA will react with free DTT rather than the reduced cystiene.
@Leo Phillips:Sir routinely in our lab we alkylate the proteins during gel digestion protocol.This is the first time i am trying to reduce and alkylate before loading gel because literature suggests that it is better to reduce and alkylate the proteins before running gel.So after running the gel i followed the same previous gel digestion protocol we use in the lab.So i want to make sure my samples are not affected.Yeah logically i do understand that one reduction and alkylation should do the job.Thank a lot Sir.
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