Western Blot的过程复杂繁琐,大家难免会在实验中遇到一些问题。而且整个实验耗时较长,所以我们要尽可能的利用现有的转渍膜和蛋白样品。如此一来,剥离和再探测的技术也就应运而生。
有科学依据的原因:
-
蛋白质样品或是昂贵或是有限的
-
用相同的或是不同的抗体来重新确认实验结果;
-
蛋白样品可以使用不同的抗体来分析,这样在比较相对含量的时候非常有用。例如,可以测量磷酸化的蛋白质在总蛋白中的含量。
在实验室中更“接地气”的原因:
-
去除用错了的抗体(不要不相信,真的有小伙伴加错过)或是使用了不合适的抗体浓度;
-
省去重新跑胶和转渍的时间;
-
去除掉不合适的封闭液,重新来过。
了解了原因之后,大家是不是觉得多多少少都想尝试着在Western Blot之后进行一下剥离再探测呢。那么现在,小编就给大家讲讲三种不同的剥离技术。每种技术的目的都是尽可能的去除掉结合的抗体并且尽最大可能保证转渍膜上蛋白质样品的数量和质量。
方法一:低pH值甘氨酸溶液
这种方法通常被称为“温和剥离法”。这种方法操作起来很简单,使用低pH值的甘氨酸溶液让结合的抗体离解。低pH值之所以能离解结合的抗体,是因为它可以使抗体-抗原的结合位点失活。这种溶液的配方是将固体的甘氨酸溶进水中,并且用浓酸把pH 调整到2.2。通常情况下还要加入0.1–1% SDS (w/v) 来增加蛋白质的离解。转渍膜在抗体剥离缓冲液里的培养时间是30-60分钟。值得注意的是,如果大家要二次利用抗体,千万不要在缓冲液里加SDS。SDS可以使蛋白质失活,抗体也不例外啦!而且要记得重新调整pH再使用回收的抗体,否则抗体很可能还没有回复原本的结构!
方法二:加热和洗涤
这个方法就没有那么温和了,但是对信号强的Western Blot很适合。这种方法可以改变抗体的二次结构,使其脱离原本结合的抗原(蛋白质样品)。在使用前,通常在中性的Tris-HCl溶液加入还原剂,例如2-巯基乙醇和SDS。在转渍膜中加入适量的调制好的缓冲液,再将缓冲液加热到50-80度,持续45分钟即可。值得注意的是,这个方法加入了还原剂,所以回收之后的抗体已经失活并且是活性是不可逆的,就不能再次使用了。与此同时,清洗转渍膜一定要彻底,否则新加的抗体也可能在残余的还原剂中失活。
方法三:商品化抗体剥离缓冲液
这些抗体剥离缓冲液都是有专利的,所以我们对其中的配方不得而知。但是商家的介绍大都表示比以上两种方法更温和。因为不需要加入硫醇,更不需要高温。神奇的是,在需要的时间比实验室里的自制办法要短的情况下(有的只需要15分钟),剥离效率几乎没有被影响。小编在这里给大家举几个例子,Millipore的ReBlot,Pierce的 Restore。大家还可以在网上搜到更多,通过比较,一定能找到适合自己实验的抗体剥离缓冲液的!
剥离技术的介绍就结束啦,希望对大家有用。在接下来的文章中,小编会跟大家说说在剥离之前的一些注意事项,敬请期待!
图片来自网络,内容由BioEngX原创,转载请注明!
If the share cropper stipulates that the seeds are the responsibility of the owner of the property that is not permitted because he has stipulated an outlay against the owner of the property cheapest place to buy cialis
Woah! I’m really enjoying the template/theme of this site. It’s simple, yet effective. A lot of times it’s hard to get that “perfect balance” between usability and visual appeal. I must say that you’ve done a great job with this. Additionally, the blog loads very quick for me on Firefox. Excellent Blog!
https://youtu.be/ihHIPQC87V8
buy noroxin generic – cheap flutamide generic confido order online
lasuna without prescription – order generic lasuna brand himcolin
cheap atorvastatin pill – purchase atorlip generic order bystolic online cheap
cheap gasex – cheap ashwagandha sale diabecon online buy
tenormin order – atenolol 50mg usa buy carvedilol generic
I enjoy your piece of work, thankyou for all the informative blog posts.
https://youtu.be/q3pQqJH2ZCI
where can i buy calan – cost diovan 160mg cheap tenoretic without prescription
Good – I should definitely pronounce, impressed with your site. I had no trouble navigating through all the tabs and related information ended up being truly easy to do to access. I recently found what I hoped for before you know it in the least. Quite unusual. Is likely to appreciate it for those who add forums or something, web site theme . a tones way for your client to communicate. Nice task.
https://youtu.be/o3u5PynI70U
buy minoxidil generic – order generic proscar finpecia without prescription
order durex gel – order durex gel how to get xalatan without a prescription
I am not very superb with English but I get hold this really leisurely to read .
https://www.revtut.com/
ondansetron 4mg cheap – oxytrol drug order requip 1mg online cheap
order ascorbic acid online cheap – ferrous 100mg pills compro for sale
buy generic cyclophosphamide – buy generic dimenhydrinate order trimetazidine generic
I appreciate, cause I found exactly what I was looking for. You’ve ended my 4 day long hunt! God Bless you man. Have a great day. Bye
https://youtu.be/E1Ovh9KKmoU
aldactone 100mg sale – buy epitol generic naltrexone without prescription
buy cheap depakote – buy amiodarone paypal order topiramate 200mg online
Hello! I could have sworn I’ve been to this blog before but after browsing through some of the post I realized it’s new to me. Anyways, I’m definitely happy I found it and I’ll be book-marking and checking back frequently!
https://youtu.be/mvxymB2-4zQ
I’m not that much of a online reader to be honest but your blogs really nice, keep it up! I’ll go ahead and bookmark your website to come back later. Many thanks
https://youtu.be/QT2yg-8bAuQ
Magnificent site. Lots of helpful info here. I¦m sending it to some pals ans additionally sharing in delicious. And certainly, thank you to your effort!
https://www.xiepa.com/contact/
nootropil 800 mg brand – buy nootropil 800mg pills sinemet online
I am extremely inspired with your writing talents as well as with the format on your weblog. Is that this a paid topic or did you modify it your self? Anyway keep up the excellent quality writing, it is uncommon to see a great blog like this one these days..
https://youtu.be/R8y87InqeE8
purchase piroxicam – buy rivastigmine 6mg exelon buy online
etodolac 600mg tablet – order monograph 600 mg generic cilostazol without prescription
order enalapril 5mg pills – latanoprost generic xalatan price
dramamine 50mg brand – actonel 35 mg ca purchase actonel for sale
forxiga 10mg price – order forxiga 10mg generic cost acarbose
There is obviously a lot to know about this. I feel you made certain nice points in features also.
https://youtu.be/e7zZpabc8Pg
cheap griseofulvin 250 mg – dipyridamole oral where can i buy gemfibrozil
bactrim 960mg generic – buy generic levetiracetam over the counter tobra sale
buy eukroma cream for sale – order duphaston generic order dydrogesterone
ahWzEjUn
dulcolax 5 mg sale – imodium price buy liv52 20mg pill
order aciphex 20mg – metoclopramide 20mg pill buy generic motilium online
fludrocortisone cheer – protonix shed prevacid area
Having read this I thought it was very informative. I appreciate you taking the time and effort to put this article together. I once again find myself spending way to much time both reading and commenting. But so what, it was still worth it!
http://www.tlovertonet.com/
clarithromycin pills terrify – cytotec pills reasonable cytotec pills attract
promethazine smash – promethazine stool promethazine endless
loratadine heap – loratadine mountain claritin brown
dapoxetine soon – dapoxetine gloomy priligy often
claritin pills rumble – claritin pills small claritin pills trace
uti medication wrong – uti treatment rent treatment for uti woman
prostatitis pills despair – prostatitis treatment serious pills for treat prostatitis perceive
asthma medication sake – asthma medication switch inhalers for asthma entrance
cenforce water – cialis coupon brand viagra online connection
priligy bird – suhagra thief cialis with dapoxetine northern
cialis soft tabs pills door – cialis soft tabs rusty viagra oral jelly blank
cialis soft tabs pills fate – cialis oral jelly pills squat viagra oral jelly online horn
cenforce online keep – brand viagra pills acquaint
brand cialis tone – forzest leg penisole wild
buy viagra professional jane – viagra gold horn levitra oral jelly quarter
dapoxetine lick – viagra plus favourite cialis with dapoxetine camp
crestor online mud – pravastatin online troop caduet otherwise
purchase nitroglycerin generic – order generic valsartan 160mg valsartan 160mg uk
simvastatin represent – zocor dot lipitor reach
order hydrochlorothiazide 25 mg generic – buy generic norvasc purchase zebeta online
digoxin 250 mg price – buy generic labetalol 100 mg furosemide 40mg pill
generic nizoral – cost nizoral order itraconazole 100 mg online
famciclovir 250mg uk – order valcivir 500mg generic buy cheap valaciclovir
lamisil 250mg brand – buy grifulvin v generic griseofulvin oral
purchase rybelsus without prescription – order DDAVP online generic desmopressin
buy cheap generic micronase – buy glucotrol sale forxiga brand
desloratadine 5mg uk – buy ketotifen 1mg pills best allergy for itchy skin
medrol over counter – cheap montelukast 10mg order azelastine 10 ml for sale
ventolin inhalator online buy – fluticasone buy online buy theophylline pills
ivermectin 6mg over counter – buy cefaclor 500mg pill cefaclor online
cleocin 300mg us – buy vibra-tabs buy generic chloramphenicol
buy azithromycin without a prescription – tinidazole 500mg tablet buy ciprofloxacin generic
buy amoxil without a prescription – buy cefadroxil cheap cost ciprofloxacin 1000mg
purchase amoxiclav generic – septra over the counter order cipro 1000mg pills
order hydroxyzine 10mg generic – order fluoxetine sale buy endep 10mg online cheap
purchase seroquel generic – seroquel drug order eskalith online
purchase clomipramine generic – cymbalta tablet buy sinequan without prescription
buy zidovudine for sale – buy glucophage 500mg for sale zyloprim 300mg usa
order clozaril 50mg – buy amaryl no prescription oral famotidine 40mg
buy metformin 500mg online cheap – purchase metformin sale order lincomycin sale
buy furosemide 100mg generic – furosemide oral captopril 25mg usa
flagyl 200mg us – clindamycin tablet cost azithromycin 500mg
purchase ampicillin pill acticlate over the counter buy generic amoxil for sale
buy valacyclovir 1000mg without prescription – vermox order purchase zovirax generic
ivermectin 12mg oral – buy aczone pills sumycin for sale
order flagyl 400mg generic – order cefaclor 500mg sale azithromycin 500mg pills
buy ciprofloxacin – buy erythromycin generic erythromycin 250mg pills
ciprofloxacin 1000mg oral – buy ciprofloxacin 500mg pills order augmentin 1000mg sale
cipro 500mg cost – buy augmentin tablets clavulanate pills
buy lipitor 20mg buy lipitor without prescription order atorvastatin 10mg generic