Hai,

There are 3 substances namely; N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) are commonly used for protein thiol alkylation. Among these iodacetamide is suitable for 2D-DIGE (Two-dimensional difference gel electrophoresis) in saturation labeling applications: the intrinsic neutral charge does not alter the pI (isoelectric point) of the biomolecule after labeling on the cysteine groups.

What kind of samples are you using??

Composition for Equilibration buffer 1&2

Equilibration buffer 1

6M Urea, 0.375 M Tris-HCL (pH 8.8), 2% SDS, 20% glycerol, 2% DTT

Equilibration buffer 2

6M Urea, 0.375 M Tris-HCL (pH 8.8), 2% SDS, 20% glycerol, 2.5% iodoacetamide.

Hi Parijat. DTT followed by acrylamide (reduction / alkylation) would be a good alternative. I use 6M Urea, 0.375 M Tris-HCL (pH 8.8), 2% SDS, 20% glycerol, 2% DTT for the first 10 min of equilibration and then 6M Urea, 0.375 M Tris-HCL (pH 8.8), 2% SDS, 20% glycerol, 2.5% acrylamide for 10 more min. It works really well in respect to reduction / alkylation. Good luck!

Let's just be clear, iodoacetamide, is an alkylating agent. Some of the alternatives have already been suggested. DTT is a reducing agent, used prior to alkylating. In the case above, the alkylating agent will be the acrylamide (so you get Propionamide).

Hope that helps.

Hai Tatiana, i have a question ??? Do you think that DTT is a good alternative to Iodoacetamide? For EQ buffer I we are using DTT and for EQ buffer II iodoacetamide as per the manufactures instructions. But you mentioned that 2.5% acrylamide for EQ buffer II... As per your comments, if we use the DTT for EQ I what is the difference between EQ I & II??? Kindly let me know...

Hai Parijat, For my experience, sometimes the problem may be the tissue homogenizing buffer.I have used rat lens and also human lens but i had a good results in rat lenses than in human lenses. here the problem was homogenizing buffer.

So please check out the suitable homogenizing buffer for your sample.

Best Wishes

Thanks to all.

@Isai Mathivanan- I have neither N-ethylmaleimide (NEM) nor iodoacetic acid , so I think I will better go for acrylamide. But in the manufactures instruction it it written EQ buffer 2 can be omitted by 10-15 minutes extra incubation with EQ buffer 1. Did you tried this? Does it work?

@Tatiana Rogasevskaia- Thanks for the valuable suggestion! I almost completely forgot about acrylamide!

@Peter Hains- Yes, I will try with acrylamide. Thanks for you valuable insight,

Regards

Parijat

Hi Parijat,

to jump in again. If you only reduce your samples (Eq1), then you'll break any disulphide bonds (S-S) present between Cys residues in your proteins. If you don't alkylate (with iodoacetamide, NEM, acrylamide etc, present in Eq 2), then the S-S bonds may reform. This is unlikely, but possible.

I have another question regarding 2D. How long do you generally run the first dimension? In sample do you use bromophenol blue to trace in first dimension? Or when the end voltage reaches according to the strip length , then you stop?

no, don't use bromphenol in a first dimension. You just have to use standard protocol, suited for the length and pH range of your strip. Once the run is complete, simply proceed with second dimmension.

You can have a look at: those:

1. Hoving S, Gerrits B, Voshol H, Müller D, Roberts RC, van Oostrum J.

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range

immobilized pH gradients. Proteomics. 2002 Feb;2(2):127-34. PubMed PMID:

11840558.

2. Hoving S, Voshol H, van Oostrum J. Towards high performance two-dimensional gel electrophoresis using ultrazoom gels. Electrophoresis. 2000

Jul;21(13):2617-21. PubMed PMID: 10949138.

I used to run my 2D according to the protocols of these guys, and the results were perfect.

Hi Parijat,

Time for the first dimension depends on the sample you run. I run first dimension until 37,500 Vh for 7cm strips. In my experience, it takes about 12 to 14 hours.

Hi Isai,

I use DTT as a reducing agent (10 min incubation) and acrylamide as an alkylationg agent (10 more minutes). I prepare 6M Urea, 0.375 M Tris-HCL (pH 8.8), 2% SDS, 20% glycerol as my equilibration buffer and just prior to incubation I add either DTT (2%; or 0.04g per 2 ml of the buffer) or acrylamide (2.5%; or 0.125ml of 40% acrylamide per 2 ml). Hope it makes sense.

@Tatiana Rogasevskaia : Are you sure about "37,500 Vh for 7cm strips" ? What pH range you were using for 7cm strips?

@Daniel Kay: I do not have iodoacetamide. Thats the only problem.

Hi Taiana

Many thanks for the information.

Hi Parijat,

For the one dimension, "10,000 Vh" for the 7 cm strip and the Ramp is Rapid. I have used 40,000 Vh for 17 cm and the pH 5-8 (narrow) range for alphaA crystallin.

Chloroacetamide is also a good alternative to IAM, you can use it at the same final concentration than IAM

Hi Parijat,

Yes, I use 37,500Vh for 7 cm strips; pH range 3-10. It works perfect for the mammalian and invertebrate membrane and cytosol proteins. The ramp takes about 2-3 hours (until reaches 4000V).

Tatiana Rogasevskaia , I m confused . in bio rad proten ief cell manual they are recommending for 10,000vh , rapid ramp. and end voltage 4000v.

I really don't know what volt hour means. can you please explain that? do I stop my run when end voltage is 4000 v? then why did you say it takes 12-14 hr? what other steps are there?

Hi Parijat

Ohhh!! You just follow the BIO-RAD manual, no confusion...... I hope Tatiana mentioned 12-14 hrs for EQ step because 17 cm strip took 5 hrs for IEF step.

Hi Daniel,

I'm afraid I'm going to have to disagree with you there. Alkylation with acrylamide, in my experience, is excellent. I'll back this up with published comparisons of acrylamide with iodoacetamide;

1. Reiko Mineki, Hikari Taka, Tsutomu Fujimura, Mika Kikkawa, Noriko Shindo, and Kimie Murayama. In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteomics 2 (12):1672-1681, 2002.

2. Takeshi Yokono, Reiko Mineki, Hikari Taka, Hiroto Kotaniguchi, and Kimie Murayama. Improvement of Automatic In-Gel Digestion by in Situ Alkylation of Proteins. Journal of Biomolecular Techniques 14 (3):191-196, 2003).

Hi Parijat,

It takes 2 to 4 hours to reach 4000V (manufacturer claims 2 hours; in reality it depends on the sample; some samples take 4 - 5 hours). At this point, the run is steady, accumulating 4000 Volts per hour. Let's say the ramp takes 2 hours; in this scenario it will take 11 hours to reach 40,000Vh. Does this make sense?

@Tatiana: Perfectly makes sense.thanks..