D*****a
发帖数: 810 | 1 CVS (9/27-10/3):
Ivory Bar Soap (3 pk) - $0.99
Use $0.50/1 Ivory Bar Soap (3 pk) printable
Final Price: $0.49
First Degree Burn Cream 0.75 OZ - $5
Pay $5, Get $5 ECB (limit 1)
Final price: Free
Buy (2) Summer’s Eve Lavender Night-Time Cleansing Bar - BOGO@ $3.79
Use (1) $2/2 Summer’s Eve Lavender Night Time product printable
Final Price: $0.90 each
Pampers Sensitive Wipes 3-pack (168 ct) – $2.99 (sale price, YMMV)
Use $1/1 Pampers Wipes 168 ct or higher printable
Final price: $1.99... 阅读全帖 |
|
D*****a
发帖数: 810 | 2 cvs (10/11 -10/17):
Free YORK Peppermint Pattie from savingstar (exp 10/13)
Offer: buy (2) select pain relief products, get $5 ECB
Buy (2) Hisamitsu Salonpas-Hot Capsicum Patch 1ct - $2.49
Pay $4.98, Get $5 ECB
Final price: Free
Buy (1) Hisamitsu Salonpas-Hot Capsicum Patch 1ct - $2.49
and buy (1) Salonpas Large Pain Relieving Patch, 4ct - $2.99
Pay $5.48, Get $5 ECB
Final price: $0.24 each
Offer: buy $10 select Colgate products, get $5 ECB (limit 2)
Buy (3) Colgate Total Daily Repair Tooth... 阅读全帖 |
|
b*y
发帖数: 946 | 3 也可以买这个
90-Count Finish Powerball Dishwasher Detergent Tablets
Amazon.com has 90-Count Finish Powerball Dishwasher Detergent Tablets (Fresh
Scent) for $14.54 - 25% coupon-10 |
|
D*****a
发帖数: 810 | 4 CVS (12/13-12/19):
Total Home Dish Liquid (10.3 oz) - $0.89
Pay $0.89, get $0.89 ECB (limit 1)
Final Price: Free
Buy (3) American Greeting Cards - $0.99
Use (1) $2/3 American Greetings Cards CVS coupon
Pay $0.97, get $2 ECB
Final Price: $1.03 Money maker
Crest Pro-Health Advanced Mouthwash - $4.99
Use $1.50/1 Crest Pro-Health Advanced Mouthwash printable
Pay $3.49, get $2 ECB (limit 2)
Final Price: $1.49
Buy (2) L’Oreal Revitalift Cleanser or Towelettes ... 阅读全帖 |
|
D*****a
发帖数: 810 | 5 CVS (1/17-1/23):
Buy (4) Irish Spring Body Wash 15oz - 2/$7
total = $14
Use (4) $1/1 Irish Spring Body Wash printable
Pay $10, get $6 ECB for buying $12
Final price: $1 each
Buy (3) American Greeting Cards - $0.99
Pay $2.97, get $3 ECB for buying 3 (limit 1)
Final price: Free
Nuper-Cainal Ointment 2 oz - $11.99
Use $10/1 Nuper-Cainal Ointment printable
Pay $1.99, get $5 ECB (limit 3, monthly deal)
Final Price: $3.01 money maker
Buy (3) Aquaphor Baby Healing Ointment (3 oz) - $7.99
total= $... 阅读全帖 |
|
D*****a
发帖数: 810 | 6 CVS (3/20-3/26):
Offer: buy (1) Crest or Oral-b multipacks (excluding toothpaste), get $5 ECB
(Limit 2)
Buy (2) Oral-B Indicator Contour Clean Toothbrush Value Pack (2 ct) - $4.99
Use (1) $1.50/2 Oral-B Adult Manual Toothbrush printable
Pay $8.48, get 2 X $5 ECB for buying 1
Final Price: $1.52 money maker
Buy (1) Oral-B Indicator Contour Clean Toothbrush Value Pack (2 ct)- $4.99
Use (1) $0.50/1 Oral-B Adult Manual Toothbrush printable
Pay $4.49, get $5 ECB for... 阅读全帖 |
|
D*****a
发帖数: 810 | 7 CVS (4/3-4/9):
Scope Outlast Mouthwash (16.9 oz) - $3.19
Use $0.75/1 Scope Mouthwash printable
Pay $2.44, get $3 ECB for buying 1 (limit 2)
Final Price: $0.56 money maker
Colgate Total Daily Repair Toothpaste (4-4.2 oz) or Colgate Max or 2-in-1 (4
.6 -6 oz) - $3
Use $0.75/1 Colgate Total Toothpaste printable
And use $2.50/1 Colgate Toothpaste CVS Coupon from Red Coupon Machine
Final Price: Free
Colgate 360 Manual Toothbrush (1 ct) - $3.49
Use $0.75/1 Col... 阅读全帖 |
|
D*****a
发帖数: 810 | 8 Seventh Generation Laundry Detergent (5 load) - $2.99
Use $2/1 Seventh Generation Liquid Laundry Product printable
And use 50% Off Seventh Generation Laundry Detergent 5 load Trial Size
Target-Cartwhee
lFinal price: $0.52 money maker
(MobiSave有 $2 cashback)
Seventh Generation Dish Liquid 22 oz - $2.99
Use 5% Off Seventh Generation Products ETS Target Cartwheel
And use $1/1 Seventh Generation Dish Liquid Product printable
Pay
submit receipt for $1/1 Seventh Generation Dish Liquid 22 oz ibo... 阅读全帖 |
|
q*******n
发帖数: 1334 | 9 多谢！这个有DPCI么？
Seventh Generation Laundry Detergent (5 load) - $2.99
Use $2/1 Seventh Generation Liquid Laundry Product printable
And use 50% Off Seventh Generation Laundry Detergent 5 load Trial Size
Target-Cartwhee
lFinal price: $0.52 money maker
(MobiSave有 $2 cashback) |
|
D*****a
发帖数: 810 | 10 CVS (5/8-5/14):
Scope Mouthwash 500mL - $3.49
Use $0.75/1 Scope Mouthwash printable
Pay $2.74, get $3 ECB for buying one (limit 2)
Final price: $0.26 money maker
Buy (4) Pampers Diapers Jumbo Packs - $8.99
Use (4) $1.50/1 Pampers Diapers jumbo packs printable
Pay $29.96, get $10 ECB for buying $30
Pampers jumbo packs
Final Price: $4.99 each
Buy (2) Eucerin Daily Replenishing Lotion 16.9 oz -$10.99
Use (2) $3/1 Eucerin Body Lotion or Creme Product (2 oz. – 16.9 oz.)
prin... 阅读全帖 |
|
D*****a
发帖数: 810 | 11 CVS (5/15-5/21):
Buy (4) L’Oreal Eye Shadow Singles - BOGO 50% Off @$5.49
Use (4) $1/1 L’Oreal Paris Product printable
Pay $12.47, get $10 ECB for buying 3
Final Price: $0.62 each
Buy (3) Crest Pro-Health Mouthwash (8 oz) - $4.29
Use (3) $1/1 Crest Pro-Health Mouthwash printable
Pay $9.87, get $6 ECB for buying $12 (limit 2)
Final Price: $1.29 each
Buy (4) Huggies Jumbo Pack Diapers or Little Swimmers Swimpants - $8.99
Use (4) $2/1 Huggies Diapers printable
(or $2/1 Little S... 阅读全帖 |
|
D*****a
发帖数: 810 | 12 CVS (6/26-7/2):
Buy (2) Crest Kid’s Fluoride Toothpaste (4.6 oz) - $3.49
Use (2) $1/1 Crest Kid’s Toothpaste printable
Pay $4.98, get $4 ECB for buying two (limit 2)
Final Price: $0.49 each
Buy (2) Huggies Jumbo Pack Diapers,Pull-Ups or GoodNites - $9.99
Use (2) $2/1 Huggies diapers printable
(or $4/2 Pull-Ups or GoodNites)
Pay $15.98. get $5 ECB for buying $15
Final Price: $5.49 each
Buy (2) Dove Shampoo or Conditioner (12 oz) -BOGO 50% off @$4.99
Use (... 阅读全帖 |
|
D*****a
发帖数: 810 | 13 CVS (7/10-7/16):
Colgate 360 Manual Toothbrush (1 ct) - $3.49
Use $0.75/1 Colgate 360 Manual Toothbrush printable
Pay $2.74, get $2 ECB for buying one (limit 4)
Final Price: $0.74
Scope mouthwash 00mL- $3.79
Use $0.50/1 Scope Mouthwash (237 ml+) printable
Pay $3.29, get $3 ECB for buying one (limit 2)
Final price: $0.29
Dulcolax Product (100 ct) - $19.99
Use $3/1 Dulcolax Product printable
Pay $16.99,
Submit receipt for Try Dulcolax FREE Mail in Rebate... 阅读全帖 |
|
D*****a
发帖数: 810 | 14 CVS (9/18-9/24):
Black Forest Gummy Worms or Bears (4 oz) - $1.50
Pay $1.50, get $1.50 ECB (limit 1)
Final Price: Free
Colgate Total Toothpaste 4-4.2oz - $2.99
Use $1/1 Colgate Total, Colgate Optic White, Colgate Enamel Health or
Colgate Sensitive Toothpaste (3oz+) printable
Pay $1.99, get $2 ECB for buying one (limit 2)
Final Price: Free
Buy (2) Colgate Optic White Manual Toothbrush (1 ct) - $3.99
and buy (1) Colgate Optic White Toothpaste (4 oz) - $3.99
Use (2) $0.75/1 Colga... 阅读全帖 |
|
D*****a
发帖数: 810 | 15 CVS (3/12-3/18):
Colgate Total Advanced Toothpaste (4-4.2 oz) - $2.99
Use $1/1 Colgate Toothpaste printable
Pay $0.99, get $2 ECB (limit 2)
Final Price: Free
Hershey’s Egg or Bunny Ears (1.2-1.55 oz) or Cadbury Egg 1.2 oz, Mini Eggs
1.5 oz - $0.99
Pay $0.99, get $0.99 ECB
Final Price: Free
Buy (3) Pamper Jumbo Pack Diapers or Easy-Ups - $9.99
Use (3) $2/1 Pamper Jumbo Pack Diapers or Easy-Ups printable
Pay $23.97, get $10 ECB for buying $30 (limit 3)
Final Price: $4.6... 阅读全帖 |
|
D*****a
发帖数: 810 | 16 CVS (3/19-3/25):
Oral-B Indicator Contour Clean Toothbrush (2 pack) - $4.99
Use $0.50/1 Oral-B Adult Indicator Toothbrush printable
Pay $4.49, get $4 ECB for buying one (limit 2)
Final Price: $0.49
Arm & Hammer Liquid Laundry Detergent (32 loads) or Ultra Power Paks 21ct -
$1.98
Use $1/1 Arm & Hammer Liquid Laundry Detergent or Ultra Power Paks printable
Final Price: $0.98
Buy (2) Kleenex Facial Tissue (85 ct) - $0.99
Use (2) $0.30/1 Kleenex Facial Tissue printable
Pa... 阅读全帖 |
|
D*****a
发帖数: 810 | 17 CVS (5/14 - 5/20):
Tide Pods or Gain Flings (12-16ct)- $3.94
Use $2/1 Tide PODS 12 ct+ printable
(or $2/1 Gain Flings)
Final price: $1.94
Buy (2) Arm & Hammer Spinbrush Toothbrush (1 ct) - $5.99
Use (1) $3/2 Arm & Hammer Spinbrush Toothbrush printable
Pay $8.98, get (2x) $3 ECB for buying two (limit 2)
Final Price: $1.49 each
Buy (2) Arm & Hammer Liquid Laundry Detergent (32 loads) - 2 for $7
Use (2) $1/1 Arm & Hammer Liquid Laundry Detergent printable
And use (1) $2/... 阅读全帖 |
|
D*****a
发帖数: 810 | 18 CVS (8/6-8/12):
Colgate toothpaste (4-6 oz) - $3
Use $0.50/1 Colgate Toothpaste printable
And use $2.50/1 Colgate Toothpaste CVS Instant coupon from the red coupon
machine
Final Price: Free
Buy (2) Crest Complete, Pro-Health or 3D White Toothpaste (3.5-6.2 oz)
- 2
for $8
Use (2) $2/1 Crest Toothpaste printable
Pay $4, get $2 ECB for buying two (limit 2)
Final Price: $1 each
(Kroger mega sale, buy 5 get $5 off, Crest toothpaste $1.49 -$1.99, free
after cou... 阅读全帖 |
|
D*****a
发帖数: 810 | 19 CVS (8/27-9/2):
Colgate 360 Manual Toothbrush (1 ct) - $3.99
Use $0.75/1 Colgate 360 Manual Toothbrush printable
Pay $3.24, get $2 ECB for buying one (limit 2)
Submit receipt for $0.50/1 Colgate Manual Toothbrush Ibotta rebate
Final Price: $0.49
Buy (1) Neutrogena SkinClearing Blemish Concealer - $9.99
and buy (1) Neutrogena Moisture Smooth Lip Stick - $9.49
Use (1) $4/1 Neutrogena Face Product printable
And use (1) $2.50/1 Neutrogena Lip Product printable
R... 阅读全帖 |
|
D*****a
发帖数: 810 | 20 CVS (10/1-10/7):
Buy (2) Pop*arazzi Nail Polish - 2 for $5
Pay $5, get $4 ECB for buying two (limit 1)
Final Price: $0.50 each
Buy (4) Huggies Jumbo Pack Diapers or Pull-ups BOGO50% off @$11.99
Use (4) $2/1 Huggies diapers or Pull-ups printable
Pay $27.97, get $10 ECB for buying $30
Final Price: $4.49 each
Buy (2) Maybelline NY Colossal Big Shot Mascara -$8.29
Use (2) $3/1 Maybelline NY Colossal Big Shot Mascara printable
And use (1) $4/1 Maybelline Product CVS Coupo... 阅读全帖 |
|
D*****a
发帖数: 810 | 21 Buy (4) Persil Detergent 40oz - $4.99
Use (4) $3/1 Persil ProClean Liquid Laundry Detergent printable
Pay $7.96, get $7 ECB for buying $20
Final price: $0.24 each
Crest Scope Mouthwash (33.8 oz) - $4.79
Use $1/1 Crest Mouthwash Manufacturer CVS App-Only coupon
Pay $3.79, get $3 ECB (limit 2)
Submit receipt for $0.50/1 Scope Mouthwash Ibotta rebate
Final Price: $0.29
Buy (4) Eucerin Intensive Repair Lotion (5 oz) - $5.79
Use (4) $3/1 Eucerin Body Product printable
Pay ... 阅读全帖 |
|
D*****a
发帖数: 810 | 22 CVS (12/17-12/23):
Purex Laundry Detergent (43.5-50 oz) - $2.99
Use $1.50/1 Purex Liquid Laundry Detergent printable
Pay $1.49, get $1.50 ECB
Final Price: Free
Buy (3) Rimmel ScandalEyes Soft Kohl Eyeliner - $4.39
Use (3) $3/1 Rimmel Eye Products printable
Pay $4.17, get $4 ECB for buying $12
Final Price: $0.06 each
Buy (2) Bounty Paper Towel (12 rl) or Charmin 16 rolls or Mega 8 rolls - $9.
99
Use (2) $1/1 Bounty Paper Towel printable
(or $1/1 Charmin Toilet Pa... 阅读全帖 |
|
D*****a
发帖数: 810 | 23 cvs (2/11-2/17):
Colgate Max Toothpaste (6 oz) or 2 in 1 Toothpaste 4.6 oz)- $3
Use $2.50/1 Colgate Toothpaste CVS coupon from the Red Coupon
Machine
Final Price: $0.50
Buy (2) Physicians Formula Eye Makeup Remover Lotion (2 oz) - $4.99
Pay $9.98, get $10 ECB for buying 2 (limit 1)
Final Price: Free
Buy (2) Aveeno Active Naturals Daily Moisturizing Lotion (2.5 oz) - $4.19
Use (1) $2/2 Aveeno Products printable
Pay $6.38, get $5 ECB for buying 2 (limit 1)
Final Price: $0.69... 阅读全帖 |
|
c*******7
发帖数: 3763 | 24 赫赫，民事的诉讼，沿海受污染的那些渔民、经济团体、旅游餐饮等经济行业，一个一
个的告起来，或者集体诉讼，就够bp喝几壶的了。
还不算环保团体、政府部门的罚款。。。
不知道已经倒了多少detergent下去了，俺就不信这么大量的detergent会无害？ |
|
发帖数: 1 | 25 Dr. Harminder Dua
Posted on March 1, 2014 by vikrant panchal
Dr. Harminder Dua
Dr. Harminder Dua is an Indian-British medical doctor and researcher.
Introduction:
Dr. Harminder Dua, born in Jalandar, Punjab.
He is an Indian-British medical doctor and researcher (with MBBS, DO, DO (
Lond), MS, MNAMS, FRCS, FRCOphth., FEBO, MD, PhD).
He is the chair and professor of ophthalmology at University of Nottingham.
He is the head of the Division of Ophthalmology and Visual Sciences.
Dua is also the co-ed... 阅读全帖 |
|
c*******y
发帖数: 3008 | 26 WALMART 有种zote的洗衣皂， 挺好用的， 1块钱14.1oz一大块儿。 就在卖laundry
detergent的架子上层。
婴儿洗衣服最好用ALL牌或dreft牌的laundry detergent， 不伤皮肤。 zote洗衣皂也
可以用 （不知道sensitive skin baby的衣服能不能用）。
chinatown canel street上的大家发 richwell里有扇牌肥皂， 有点贵。 |
|
j**4
发帖数: 10425 | 27 en, some washer says it has to use high efficiency detergents. it is
claimed that 1 cup of he, equals 2 or 3 cups of normal detergent. but i am
sure if u put in non he, the washer will be fine |
|
e**********e
发帖数: 999 | 28 CHD
Church & Dwight Co., Inc. develops, manufactures and markets a range of
household, personal care and specialty products. The Company’s brands
include ARM & HAMMER, (used in multiple product categories, such as baking
soda, carpet deodorization and laundry detergent), TROJAN Condoms, XTRA
laundry detergent, OXICLEAN pre-wash laundry additive, NAIR depilatories,
FIRST RESPONSE home pregnancy and ovulation test kits, ORAJEL oral
analgesics and SPINBRUSH battery-operated toothbrushes. The Compan... 阅读全帖 |
|
s******r
发帖数: 5309 | 29 没错，楼主八成觉得买便宜油对不起他的好车，纯心理作用。
全美卖的油都差不多，和谁家的加油站没有一毛关系，除了东部州都加酒精，西部的有
些不加的区别。87号的10%酒精的mileage不如85号不加酒精。有的说shell的油
detergent多，那只有对camery之类特别容易积碳的烂engine才有意义。而且detergent
每20000mile自己加一瓶就好，根本没有必要多花钱加shell的油。 |
|
w*****r
发帖数: 385 | 30 Personally I prefer Spray on. I like Grangers and Revivex, the spray allows
you to go heavier on high wear areas.
Btw:Regular Detergent takes over 100 washes to get out it is the absolute
worst thing you can do to your Jacket.
Wash in either
Nikwax Tech Wash
Grangers G-Wash
Or any NON Detergent Soap I think Zero may still qualify. |
|
j*********r
发帖数: 654 | 31 一般是不洗的，但是我洗过好几回。如果有removable frame／foam/inserts, 先要取出来，能够分离的、拆开的部件都拆开。 勤快的可以泡在浴缸里洗，用一点laudry detergent, 讲究的用nikwax 或者Granger 等牌子的outdoor clothing cleaner (it has much less residual than normal laundry detergent），然后洗衣机甩干，再过一遍水，甩干。如果不想手洗，而且包包的体积不大，可以直接机洗。
自然晾干，不能烘干。 |
|
s***n
发帖数: 10693 | 32 谢谢，我的foam好像取不出，而且45升体积不小了塞不进洗衣机，凑合用一阵再说吧。
:(
取出来，能够分离的、拆开的部件都拆开。 勤快的可以泡在浴缸里洗，用一点laudry
detergent, 讲究的用nikwax 或者Granger 等牌子的outdoor clothing cleaner (it
has much less residual than normal laundry detergent），然后洗衣机甩干，再过
一遍水，甩干。如果不想手洗，而且包包的体积不大，可以直接机洗。 |
|
j*********r
发帖数: 654 | 33 有救。
You should have followed the washing instruction labels. Do NOT wash them
using "normal" liquid detergent. It was the surfactant in the "normal"
liquid detergent that destroyed the DWR coating.
From now on, use this one or similar products:
http://www.rei.com/product/794731/grangers-one-step-wash-and-wa
and Follow the instructions!!!
Granger products are a bit cheaper than Nikwax and pretty much the same
stuff.
Nikwax is worth buying if it is 50% off. See here:
http://www.sierratradingp... 阅读全帖 |
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p**********9
发帖数: 3463 | 34 Washer/dryer can do a better job. But I don't use regular detergent, I use
detergent for sensitive skin,(Tide Free), always rinse them for extra time. |
|
w*******e
发帖数: 1588 | 35 Using shampoo as alternative detergent. Silk is mainly of protein, which
prefers neutral detergent. Hand wash in cold water should be good for all
silk, unless it's lined or the fabric is not pre srunk. If the silk fabric
is not ore-shrinked, it may shrink a little after wash. |
|
l*****t
发帖数: 77 | 36 Sorry no Chinese now.
Use dish detergent or laundry detergent? Thanks |
|
b*s
发帖数: 82482 | 37 HE 是 High Efficiency
Don't use your regular Tide. Now for the reason WHY: All front loading, and
a few top loading, washers, use small, high speed, water pumps. When these
pumps are called on to pump, the very small pump will turn very fast trying
to move as much water as its much larger, older cousins. If there is even
the slightest amount of SUDS-MAKING detergent in the water, the small pump
will instantly turn the water to suds, thus locking down the pump. The
washer will sense this lock down... 阅读全帖 |
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d********3
发帖数: 7220 | 38 呵呵难怪这么多中国人不爱用洗碗机，偏见多么容易养成，又多么容易被坚持。老的洗
碗机确实是费水费电，噪音还大，就不讨论成本问题了。
现在新出的洗碗机哪个不是省水省detergent啊，你想那么一大堆的碗，分十次手洗难
道用的detergent比那一小块会少？我家用的是Cascade带一点液体的洗碗块，洗出来的
碗绝对比手洗的干净。
我和老婆原来2个人也用洗碗机，大概3、4天才洗一次，就多买些碗碟好了，一点没觉
得不方便。
洗碗机用热水多点那是肯定的，但是还兼顾了消毒呢，人工什么就不算在内了，如果这
么多人觉得洗碗机浪费能源，那为什么还用洗衣机烘干机呢，直接用手洗得了。。。偏
见啊偏见 |
|
f******w
发帖数: 10267 | 39 没有detergent之类，那上面写了detergent和fertilizer不能运。
, |
|
j*****a
发帖数: 658 | 40 1。 proteinase inhibitor cocktail and phosphatase inhibitors 是要加的。他们
的作用是防止你在抽提蛋白的过程中蛋白讲解。在你一开始用pbs悬细胞的时候，如果
不加这两种抑制剂，会有蛋白降解。一般我洗细胞的时候就开始加。当然蛋白煮过变性
就不存在这个问题了。
2。 你的方法当然是可行的。5x sds page loading buffer 里就有detergent SDS，然
后再加热裂解，当然没错。如果你对自己的手平行操作非常confident，是完全可以不
测蛋白浓度的，直接加热完后run gel。 其实测蛋白的时候如果做的好一般情况下，
sample和sample之间浓度都是差不多的，校正和不校正是没有很大差的。所以你的做法
没有啥不对。只是你老板的做法是小学生式的，step by step, 也是classic的做法。
3. RIPA buffer是很强的裂解液，里面有几种detergent 组合，所以一般情况下裂解是
很完全的。就像上面的人说的，超声主要是打碎DNA，有点牛刀杀鸡了。
4。关于loading contro |
|
m****m
发帖数: 395 | 41 The reason why I didn't dialysis is that my protein is a hydrophobic
membrane protein which is desolved in an expensive detergent. If I dialysis,
the detergent will also go away and my protein will precipitate anyway.
I wonder if there's any other way to concentrate protein except using this
method and centrifuge through a membrane to get rid of the water? The later
is not so efficient, because I see some protein also pass the membrane which
causes loss.
Can we use vaccum dry under low temperatu |
|
s******y
发帖数: 28562 | 42 这几种方法你都可以试一下。没有哪一个是肯定保险的。
至于说用concentrator会损失蛋白？这个可能是因为你没有选对大小合适
的滤膜。一般来说，滤膜的孔的标定尺寸应该比你的蛋白的真正尺寸小
三倍。比方说30K 的蛋白必须用小于10K MWCO的膜，否则蛋白有可能漏过去。
另外，在dialysis 的时候，你可以在外面的溶液里也放上detergent 啊，
我不明白这个为什么会是问题。而且，一般来说，detergent 在溶液里
会形成微团，所以不会容易的透过滤膜
dialysis,
later
which |
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y****l
发帖数: 1 | 43 大家好，我最近拿到了一个十二个跨膜结构膜蛋白的晶体，正在做SeMet标记晶体。
我用的菌株是：B834（DE3）
培养基：M9+19个氨基酸+0.5%glycerol+ 5%glucose +Thiamine+1mM MgSO4+0.1mM
CaCl2+0.05mg/ml selenomethionine+amp
温度：37°
培养时间：24小时
用这样的方法，没4L菌可以大概拿到3毫克的蛋白。
现在问题是3毫克蛋白根本不够我用来长晶体。
问题：
1.在养细胞的时候我发现离心以后上清还是很浑浊，还有大量的菌在上清中无法被离下
来。我
加大了离心的速度和时间，还是无法离下来。有好多菌附着在离心管壁上。不知道这是
为什
么？
2.离下来的菌有一些黄黄的东西，而且是比较多的。通过我的纯化，我发现这些黄黄的
东西是
不能被detergent溶解的，里面没有我的目的蛋白。不知道这些黄黄的东西是什么？
3.离下来的菌在悬在buffer的时候，我发现很粘稠，和我用与表达非标记蛋白用到的菌
的时候
是完全不一样的。是不是菌破裂了，dna跑了出来。很奇怪。
4.长SeMet derivative pro... 阅读全帖 |
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s******u
发帖数: 81 | 44 多谢！
另外的原因可能还有digestion enzyme的sequence preference.
说到co-IP, lysis buffer一般都加detergent吗？看别人的文章基本都加，可有的
interaction加了detergent几乎检测不到。
stringency |
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c********b
发帖数: 363 | 45 每个步骤都是别人都知道的,我只是组装了一下. 最大不同是别人是先Urea变性,提出来
再复性.我是利用Triton X-100可以洗脱SDS的特点利用SDS变性,然后Triton X100洗脱
复性,然后过柱. 肯定不会对所有蛋白适用,但是起码我遇到的都work (包括一个>100
Kd的酶).
我是用的sonication water bath, 也可以用probe sonication再加SDS,都OK,但是注意
dilution的比例和体积.
Good luck. 用的高兴分我几个包子.
Key, keep a fraction from every step for analyses and trouble shooting
- Culture the E. coli overnight
- Induce it at a proper concentration of IPTG, room temperature for ~
4hrs
- Pellet the E. coli at 6000 rpm at 4 degree
- R... 阅读全帖 |
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b******y
发帖数: 627 | 46 High salt can help eliminating non-specific interactions through salt
bridges. Adding some detergent, say 0.02% NP-40 or something alike, can help
eliminating non-specific interactions through hydrophobic interactions.
If affordable, I would wash three-five times with high salt with detergent
buffer. |
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l***y
发帖数: 4671 | 47 不加 SDS 的话，电泳主要靠蛋白质自己的电荷量，还要保证负电荷为主，需要 pH 高
于所关心的蛋白质的等电点。带的位置和蛋白质大小也没有严格的关系，要靠
antibody 来标记。同时因为电荷量不够，蛋白质会 aggregate，所以对 detergent 的
浓度很敏感。
SDS 主要靠长烷基链的憎水性来和憎水的蛋白链结合，所以会 denature 掉蛋白质，破
坏靠范德华力维持的 protein association。可以考虑用一系列浓度不同的低浓度 SDS
处理 sample 跑电泳，看 dimers 带的变化，做曲线再外推。这个方法还是对
detergent 的浓度特别敏感。而且外推的曲线很不直。。。
还可以考虑一些不会显著 denature 蛋白质的引入负电荷的办法，比如说 Coomassie G
-250 据说只会在 protein 表面引入负电荷。一直想试。哪位试过，谈谈感受？ |
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s*******e
发帖数: 1010 | 48 估计人家想问的是怎么不用detergent就纯化到了膜蛋白，潜台词是你怎么保证膜蛋白
在无detergent体系中保留了正常功能和天然构象。 |
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z*********8
发帖数: 1203 | 49 可是detergent本身也有灭菌作用啊，提细菌dna的时候不都加点detergent么？这篇文
章怎么看起来有些熟悉的感觉啊！ |
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s******y
发帖数: 28562 | 50 Polysome purification and fractionation
(Based on Davies & Abe, 1995, Methods in Cell Biology, Vol 50, Chapter 15)
1. Prepare 4.6ml 15-60% sucrose gradients in 5ml polyallomer tubes (Beckman
#326819) by
pipetting 2.3ml of 60% sucrose in buffer B followed by 2.3ml of 15% sucrose
in buffer B
(slowly, drop by drop, against the wall). Cap the tubes with rubber stoppers
and lay tubes
horizontally for 3h @RT for sucrose to diffuse (or for 2h if making 10-40%
gradient). Place the
tubes again vertically... 阅读全帖 |
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