Q:1,Hi !I have been trying to clean and concentrate proteins form eucariotic Cell culture medium in order to analyse them in 2D. I’ve read some papers were TCA precipitation was used and recommended for this purpose but I have not been able to redissolve my samples. I have used 10%TCA and let it precipitate for 30 minutes in Ice, afterwards I have centrifuge the sample, washed it with diethil ether and tried to dissolve the pellet in ordinary IEF sample solution containing Urea, Chaps, ampholites and DTT. I wasn’t able to dissolve the sample even using a 9M Urea concentration.
Do you have any suggestions on how to dissolve proteins from TCA precipitation or an alternative way to clean and concentrate proteins from culture medium for 2d analysis?
A:This problem occurs very often.
Better for cleaning and concentration of protein samples: Use the Ettan 2-D Cleanup Kit from Amersham Bioscience s
其实真核细胞总蛋白质的提取用不着使用TCA/acetone法,细胞相对于植物 组织还是比较干净的,少掉了一些脂类酚类杂质.对于组织来讲,使用clean-up kit效果还是不错的
Q:Can anyone help with a recent problem with proteins only running half way despite the running dye reaching the end. This problem has suddenly appeared, running buffer is new batch but has been pH"d and made to the same recipe as before. Gels are 10%T being run under the same conditions as always (70V overnight at RT). Not only do the proteins on the gel seem to disappear towards the bottom half but the molecular weight markers also become very faint and blurry. It almost looks as if the low Mw proteins have somehow diffused out of the gel, although this does not seem possible. I know that I cannot expect low Mw proteins to focus as sharply as high, however they have never been this fuzzy before.
A:Never "pH" the running buffer!! In this way you have introduced Cl- Ions into the Tris-glycine buffer: this is a No-No. The system has to move all Cl-ions out of the cathodal buffer tank before glycine and proteins start to migrate. When you make your running buffer, just use Tris, glycine and SDS of good quality, according to the stsndard recipe. You do not need to measure the pH; and you must not titrate the running buffer!
codegreen点评:兄弟们知道这个同志犯了什么低级错误吗,他老人家自做聪明使用HCL去调Tris-Glysin running buffer的PH值,危害是电泳系统在开始迁移甘氨酸和蛋白质之前必须把阴极的CL离子都跑出去,结果自然就造成了蛋白质斑点迁移的滞后了,呵呵.
Q:Why is it not necessary to use a reducing agent such as DTT when samples are being subjected to isoelectric focusing with the destreak reagent? I am just concerned that if disulfide bonds are present in the protein mixture, the destreak reagent itself will not be able to reduce them. It seems that it would be important to add DDT.
A1: Destreak is a reductant and a disulfide. It is actually hydroxyethyl disulfide or HED. It is very similiar to another popular reductant, 2-mercaptoethanol; HED is basically what 2-ME would be if it formed a disulfide bond with another 2-ME molecule. Having too much DTT around will break the disulfide bonds made with HED. The big benefit of DeStreak (HED) is that when it is in a disulfide bond it is essentially non-ionizable where as DTT still has an ionizable group in the form of the other thiol group. (I would not recommend using 2-ME as a reductant as it is usually badly contaminated with impurities)
A2:You extract your sample with a reductant, like DTT. This reduces the disulfide bonds. This sample is loaded via cup-loading at the anodal side on the IPG strip, which was pre-rehydrated with urea, thiourea, detergent, IPG buffer and DeStreak
点评: DeStreak (HED)试剂是一种新型的还原剂,非常近似于2-巯基乙醇.在上样缓冲液中加入DTT过多(通常是超过10mM)会严重干扰DeStreak (HED)的还原效果.因此,建议使用DeStreak (HED)时最好采取上样杯加样,提取样品时仍使用DTT,而使用DeStreak (HED)进行IPG胶条的泡涨.这样,最终的DTT浓度稀释到了10mM以下.
Q: I want to keep 2-D gels for analyze it latter, with out the risk of damaging proteins quality and/or gel freezing. Is DDH2O with 20% Glycerol in -20oc is o.k.?
A1: Don"t freeze the gel. Store it at 4C in a plastic bag. I would also make sure that you soak the gel in something with about 10% ethanol so you don"t get microbial growth. A 20% glycerol solution is a very good growth medium.
A2: Even if you later decide very much later that you want a peptide mass fingerprint from the dried gel it can be done. I returned to a gel that I had left dried in an old lab book for 8 years. Once we had a Maldi-tof MS and a genome project had rolled by I was able to return to an ancient band that was all that was left of my tediously purified protein and find its gene from its peptide mass fingerprint. (See Insect Biochem. Mol. Biol. 31 (6-7): 513-520, 2001.)
I"ve been running 2D gels for over a year now. Most of the time my gels look good or OK, except for the spots in the acidic area. These spots are not very well resolved and give me smear. Occasionally I also get vertical distortions on my gel which I attribute to an air bubble trapped between the strip and the gel. I use Amersham"s 12.5% gels and Ettan DALTtwelve apparatus. I work with rat liver nuclear extracts that I prepare for IEF using Amerasham"s PlusOne 2-D CleanUp kit. Gels are stained with SYPRO Ruby.
Last week I ran 3 gels simultaniously. Two of the gels looked good, although the third gel, in addition to the usual streaks in the acidic area, had an ugly-looking basic area (right part of the image attached). After the IEF, the strip was normal in appearance, so I believe that the problem originated during the second dimension run. What could cause the basic area look so distorted? I want to avoid this problem in the future.