1,關于使用三氯醋酸沉淀法后蛋白質難于重懸的問題
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?
Thank you!
A:This problem occurs very often.
Better for cleaning and concentration of protein samples: Use the Ettan 2-D Cleanup Kit from Amersham Biosciences
點評:這個問題由AMERSHAM著名科學家回答,比較會替公司做廣告.
其實真核細胞總蛋白質的提取用不著使用TCA/acetone法,細胞相對于植物組織還是比較干凈的,少掉了一些脂類\酚類雜質.對于組織來講,使用clean-up kit效果還是不錯的
2.在第二向電泳中,為什么我的溴氛藍條帶都跑到玻璃板下緣了而蛋白質只跑了一半啊,郁悶!
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.
Thanks
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!
點評:兄弟們知道這個同志犯了什么低級錯誤嗎,他老人家自做聰明使用HCL去調Tris-Glysin running buffer的PH值,危害是電泳系統在開始遷移甘氨酸和蛋白質之前必須把陰極的CL離子都跑出去,結果自然就造成了蛋白質斑點遷移的滯后了,呵呵.
關于Destreak reagent的原理和使用
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
點評: DeStreak (HED)試劑是一種新型的還原劑,非常近似于2-巰基乙醇.在上樣緩沖液中加入DTT過多(通常是超過10mM)會嚴重干擾DeStreak (HED)的還原效果.因此,建議使用DeStreak (HED)時最好采取上樣杯加樣,提取樣品時仍使用DTT,而使用DeStreak (HED)進行IPG膠條的泡漲.這樣,最終的DTT濃度稀釋到了10mM以下.
怎樣保存2-D凝膠?
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.?
Thanks
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.)
點評:2-D凝膠通常在4度保存,置于密封之塑料袋中,可以加入10%的乙醇(小弟自己保存在1%的冰乙酸中,4度,一般放2周-1個月沒什么問題,雖然文獻報道可以放更久,但是心里有點怕,故未作冒險嘗試
),最好不要單獨使用20%的甘油,這個東東乃是細菌的良好培養基
后面第二個牛人做完干膠后放了八年!太恐怖了!八年前質譜還不夠成熟,此外尚無完整的基因組數據,所以這個牛人八年后才做的肽指紋圖譜,NB吧!
二維電泳圖譜的扭曲
Q:Hello!
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.
Thank you for any suggestions.
A:My guess: the lower edge of the gel was not completely flush with the glass plate edge of the support cassette. The gel edge, not the film edge, should be flush with the glass plate edge
二維電泳圖譜的扭曲
Q:Hello!
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.
Thank you for any suggestions.
A:My guess: the lower edge of the gel was not completely flush with the glass plate edge of the support cassette. The gel edge, not the film edge, should be flush with the glass plate edge
偶師弟曾經問的一個問題,圖比較菜,他剛開始做2-D電泳圖譜,大家不要笑他喔, (其實也有老外做的東東簡直是垃圾,我當時笑他們也好意思貼出來,然后著名科學家幽默地說您這次的圖已經比以前貼出來的好得多了)
Q:my recent result below,give some advices ,please !!! the high background of the top area may partly due to not pouring the developing solution out through the lateral hole of the dying pot in time.why are there so many vertical stripes in the top area?
spot number is a little bit less, right?my loading sample is 100microgram, and proteins are detected by silver staining.
A1:The vertical stripes are coming from too short equilibration. equilibrate two times for 15 minutes, not 10 min.
A2:Furthermore: Start your second dimension with 2.5 W / gel for 40 minutes, not 5 W / gel for 30 min: it will give you a better separation quality.
他的電泳條件:sample lysis buffer:8M urea,4% CHAPS, 0.5%IPGbuffer, (v/w=3:1)
sample stored at -70degrees
rehydration buffer: 8M urea,2% CHAPS, 0.5%IPGbuffer,20mMDTT
IEF protocol: 30v 6h, 60v 8h, 200v 1h, 500v 1h, 1000v 1h,
1000~8000V gradient 30min,
8000v 60000vhrs. (IPGphor)
Equilibration: 2×10min, DTT, IAA
2nd electrophoresis: 400v/400mA/5w/gel 30min
400v/400mA/17w/gel 3hrs (Ettan-DALT six)
silver stain: Vorum protocol
點評:他平衡的時間不夠,兩步10分鐘顯然不夠充分,SDS膠束尚未與蛋白質完全結合,可能也是縱條紋形成的原因之一.此外,好象AMERSHAM的電泳槽(尤其是ETTAN DALT SIX)推薦恒功率而BIO-RAD的電泳槽推薦恒流,不知大家有什么意見?
Q: I'm trying to use DTT(20mM) and TBP(2mM) simultaneously in my rehydration buffer,did they interfere with each other?Thanks in advance.
A: I wouldn't bother with TBP in the rehydration solution. It is good for lysis, etc., but it has a fairly short half-life so by the time rehydration is done and you are ready to focus the TBP is probably gone.
Q: So, you mean is better to use TBP only when you apply the sample after rehydratation? Only for IEF focusing?How can I manage with TBP, it is written on the Material Safety Data sheet ...spontaneously flammable in air....
A: When you do cup-loading, you should be underlaying the sample in an oil-filled cup so the sample won't evaporate and crystallize out. I would only use TBP in sample preparation/homogenization. (Yes, spontaneously flammable in the air would cause me concern too.)
點評:其實上面的那些帖子小弟實在不夠資格點評,實在讓大家見笑了.不過這一個確實需要點評一下:因為,這個問題是兩年前小弟自己問的.我當時知道了三丁基磷酸的好處(還原效果遠高于DTT,而且其最大的優點是其不帶電,這樣在等電聚焦過程中堿性端的TBP并不象帶負電的DTT那樣向正極移動,因此,堿性端保持著充足量的還原劑,蛋白質不會因還原劑不足而出現不同程度的氧化造成拖尾),決定采用,但又感情上拋棄不下DTT(慚愧ing),于是問這個著名科學家(sjouke hoving,想必做蛋白組學久一點的同行都有印象)是否能同時采用DTT和TBP,因為自己感覺多加還原劑效果應該更好啊,于是他建議說TBP是一種易自燃的物質,需要注意,并且建議僅在樣品提取時用于還原作用,但泡漲時最好不用(因為泡漲時間太長而TBP半衰期很短),故偶在后來的7-10堿性膠條2-D電泳中均采用泡脹膠條后,cup-loading時再加TBP,結果跑出來的電泳效果非常不錯.不是吹牛,堿性膠條的橫向條紋較輕,比有些人跑的3-10的還強.
這個方法吐血推薦(因為經過自己的實踐證明):希望用過cup-loading的兄弟一起探討一下,咱們的雙向電泳聯會不要成為擺設.
PS:TBP用好了真不一定比AMERSHAM吹了半天NB的destreak差.而且平衡時可以簡化步驟,一步平衡,還省iodoacetamide。如果做質譜，烷基化步驟可以放在酶切那一步。
