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Guidelines for Sample Preparation,

Continued

Method 1: Routine

Staining and

Blotting

Use this protocol if you are performing SDS-PAGE followed by routine staining

or blotting.
If the E-PAGE

™

Loading Buffer 1 (4X) is stored at 4

C, bring the buffer to room

temperature and mix briefly prior to use.
1.

Prepare your samples in a total volume of 10 µL in the E-PAGE

™

Buffer 1 (4X) as described below. If you need to prepare samples in a volume

of 5–15 µL, adjust the volumes accordingly.

Reagent

Reduced

Non-reduced

Protein Sample

x µL

E-PAGE

™

Loading Buffer 1

(4X)

2.5 µL

NuPAGE

Sample

Reducing Agent (10X)

1 µL

—

Deionized Water

to 10 µL

Incubate the samples at 70

C for 10 minutes.

Proceed to

Loading E-PAGE

™

Gels,

page 16.

Содержание E-PAGE Gels

Страница 1: ...uide General information and protocols for using E PAGE Gels Catalog Number EP096 06 Revision B 0 Publication Number MAN0000374 Publication Part Number 25 0644 For Research Use Only Not for use in dia...

Страница 2: ...For support visit www lifetechnologies com support or email techsupport lifetech com www lifetechnologies com 06 October 2021...

Страница 3: ...E Gels with the iBlot Device 25 Semi Dry Blotting of E PAGE Gels 31 Semi Wet Blotting of E PAGE Gels 35 Visualizing and Staining of E PAGE Gels 38 Visualizing Lumio Fusion Proteins 39 SYPRO Ruby Stain...

Страница 4: ...ns See page 71 for ordering information Product Quantity Catalog no E PAGE 48 8 Gels 8 pack EP048 08 E PAGE 96 6 Gels 8 pack EP096 06 Kit Contents Each pack of E PAGE Gels contain the following conten...

Страница 5: ...plications such as western blotting or staining The E Editor 2 02 software allows digital images of the E PAGE Gels to be reconfigured into a side by side format for easy comparison and analysis Appli...

Страница 6: ...y blotting page 25 and semi wet blotting may also be performed page 35 Tank blotting is not recommended but can be performed if even pressure is maintained between the gel and the membrane and cooling...

Страница 7: ...nes This configuration provides a 3 2 cm run length The wells of E PAGE 48 Gels are compatible for loading with a multichannel pipetter in alternating lanes or with an automated liquid handling system...

Страница 8: ...etter or with an automated liquid handling system See page 17 for automation specifications After electrophoresis the E PAGE cassette is easily opened with the Invitrogen Gel Knife Cat No EI9010 sold...

Страница 9: ...ls The Daughter E Base does not have an electrical plug and cannot be used without a Mother E Base E PAGE Gels are not compatible with the E Gel 96 bases Cat nos G7100 01 G7200 01 previously available...

Страница 10: ...nd cannot be connected to an electrical outlet The Daughter E Base is connected to a Mother E Base or to another Daughter E Base already connected to a Mother E Base Once connected to a Mother E Base...

Страница 11: ...ation about the E Holder Platform E PAGE Blotting Pad The E PAGE Blotting Pad is supplied with the E PAGE Gels and E PAGE Starter Kits It is necessary to use the pad during semi dry blotting to ensure...

Страница 12: ...12 Lumio Gel Sample Buffer Materials Needed The following items are needed for sample preparation See page 71 for ordering information Protein sample NuPAGE Sample Reducing Agent 10X E PAGE Loading B...

Страница 13: ...a range of concentrations to determine the optimal concentration for your sample Note To ensure a proper LDS lithium dodecyl sulfate from Loading Buffer 1 to protein ratio limit sample protein or lip...

Страница 14: ...r type of SDS PAGE buffer to prepare samples for Lumio Green detection Molecular Weight Standards The following protein molecular weight standards and loading volumes are recommended for E PAGE Gels B...

Страница 15: ...e and mix briefly prior to use 1 Prepare your samples in a total volume of 10 L in the E PAGE Loading Buffer 1 4X as described below If you need to prepare samples in a volume of 5 15 L adjust the vol...

Страница 16: ...mple Buffer 4X as the sample is already prepared in this buffer 2 Thaw the Lumio Green Detection Reagent and mix well 3 Add 0 1 L Lumio Green Detection Reagent to the protein samples from Step 1 in a...

Страница 17: ...oper setting prior to loading samples on the E PAGE 48 or 96 Gels This ensures proper loading of samples into the wells See page 17 for automation guidelines Dispose of gels as hazardous waste Avoid t...

Страница 18: ...t salt or protein concentrations in adjacent lanes E PAGE 48 and 96 Gels are not compatible with the E Gel 96 Mother and Daughter Bases Cat nos G7100 01 G7200 01 available previously from Life Technol...

Страница 19: ...ess and hold the time button to increase the time to the desired run time If the time button is not released the time setting increases until it reaches 00 To begin cycling through the numbers again s...

Страница 20: ...n the base begins running and a red light illuminates at the lower left corner of the base The digital display shows the appropriate time for a selected program or the last time setting Ready Mode Not...

Страница 21: ...esis press and release the pwr prg button located on the lower right corner of the Mother E Base Device The red light changes to a green light and the digital display shows the count down time while t...

Страница 22: ...ding of E PAGE 96 Gels The wells of the E PAGE 96 Gel are staggered to provide maximum run length see Figure 1 below For proper loading of samples it is important to program your automated liquid hand...

Страница 23: ...ifetechnologies com epage For automated loading of E PAGE 48 Gels position the plate in the Portrait orientation rather than the Landscape position as shown below This orientation is available for som...

Страница 24: ...older 1 Open the package and remove the E PAGE Gel 2 Remove comb from the E PAGE cassette 3 Place the E PAGE cassette in the E Holder Platform Align the bottom left end of the cassette in the lower le...

Страница 25: ...remove the gel from the E Base Device to check the progress of the run Then To continue the run from the point at which it was stopped reinsert the gel and press and release the pwr prg button The lig...

Страница 26: ...4 minutes for EP or last time setting Steady red Gel cassette inserted into a base Ready with no current flowing through gel Count down time Steady green Press and release the pwr prg button Run Negat...

Страница 27: ...eady mode after a manual stop Flashing ER Continuous loud beeping Press and hold pwr prg button for 2 seconds and remove gel from the base Failure EP last program used EP or EG No cassette Time increa...

Страница 28: ...s are completely separated Caution Use caution while inserting the Gel Knife between the two plates to avoid excessive pressure on the gel 3 Gently pull apart the cassette halves with your hands until...

Страница 29: ...the cassette for transfer after completion of electrophoresis as described on page 25 There is no need for any pretreatment of the gel after electrophoresis Wash the E PAGE gel briefly in deionized w...

Страница 30: ...of the iBlot Anode Stack Bottom and keep the stack in the transparent plastic tray 3 Place the iBlot Anode Stack Bottom stack with the tray to the left of the blotting surface area such that the tab o...

Страница 31: ...ontaining your protein samples on Metal Spacer 1 such that the gel is aligned to the lower right corner of the bottom stack with the wells of the E PAGE gel facing up 6 Clean the Metal Spacer 2 with a...

Страница 32: ...ray using the iBlot E PAGE Tab figure C A B C 9 Place the iBlot Cathode Stack Top without the tray on top of Metal Spacer 2 with the copper electrode side facing up and agarose side facing down Ensure...

Страница 33: ...stack surface Performing Blotting After assembling the iBlot Gel Transfer Device perform blotting within 15 minutes of assembling the stacks with the gel as described below 1 Close the iBlot Lid and s...

Страница 34: ...rocedure or stain the membrane see next page for details Note If you are using PVDF membranes place the membrane immediately into water as PVDF membranes dry quickly If the PVDF membrane is dried re w...

Страница 35: ...and a power supply Materials Needed You will need the following items See page 71 for ordering information Semi dry blotter Methanol for transfer of E PAGE 48 Gels NuPAGE Transfer Buffer 20X NuPAGE A...

Страница 36: ...Buffer Component Transfer Buffer with 15 Methanol Transfer Buffer with 10 Methanol NuPAGE Transfer Buffer 20X 50 mL 50 mL NuPAGE Antioxidant 0 5 mL 0 5 mL Methanol 75 mL 50 mL Deionized Water to 500...

Страница 37: ...n Tray PVDF 1 Use pre cut Invitrolon Filter Paper Sandwich or cut PVDF membrane to the appropriate size 8 6 cm x 13 5 cm 2 Pre wet the membrane for 30 seconds in methanol ethanol or isopropanol Briefl...

Страница 38: ...gel from the transfer buffer Gently rub a gloved finger over the well side of the gel to remove small gel pieces from the gel surface Re submerge the gel in transfer buffer to remove any gel pieces f...

Страница 39: ...aratus that can accommodate an E PAGE Gel 8 6 cm x 13 5 cm may be used However you may get sub optimal results if your blotting apparatus is prone to transfer distortion when accommodating thick gels...

Страница 40: ...ntil saturated Remove air bubbles by squeezing the blotting pads while they are submerged in buffer Note Use the blotting pads that fit the XCell II Blot Module Do not use the E PAGE Blotting Pads for...

Страница 41: ...r 6 Place two pre soaked blotting pads into the cathode core of the blot module Note Use the blotting pads that fit the XCell II Blot Module Do not use the E PAGE Blotting Pads for semi wet transfer 7...

Страница 42: ...y Background Lumio Green Detection Reagent Pre label proteins during sample preparation run the gel and visualize total time 1 hour 1 5 pmole SYPRO Ruby Protein Gel Stain Fix 0 5 h Stain 1 5 h Destain...

Страница 43: ...ize Lumio fusion proteins 1 Place the gel cassette on a UV transilluminator equipped with a camera and select the ethidium bromide or SYBR Green filter on the camera You may also use a laser based sca...

Страница 44: ...f your staining container To obtain good results the volume of solution must be sufficient to cover the gel completely and to allow the gel to move freely during all of the steps Staining Protocol 1 A...

Страница 45: ...8 acetic acid in deionized water see note below Methanol regular protocol only Ethanol microwave protocol only 2 pieces of nylon membrane microwave protocol only The volume of fixing staining and dest...

Страница 46: ...to completely cover the gel in a microwave safe container such that the gel moves freely during the staining and destaining steps 1 After electrophoresis remove the gel from the cassette page 24 and...

Страница 47: ...solutions will depend on the volume of your staining container To obtain good results the volume of solution must be sufficient to cover the gel completely and to allow the gel to move freely during a...

Страница 48: ...afeStain to cover the gel Incubate at room temperature for 1 5 hours with gentle shaking Decant the stain 4 Wash the gel with deionized water for 3 hours with intermittent water changes Protocol B 1 A...

Страница 49: ...lver Staining Kit Microwave oven 700 1000 watts 30 ethanol made with ultrapure water 100 ethanol Fixative 40 ethanol 10 acetic acid made with ultrapure water For SilverXpress Silver Staining SilverXpr...

Страница 50: ...Add 150 mL microwave for 45 seconds Agitate the gel for 1 5 hours Wash Ethanol 60 mL Ultrapure water 140 mL Final Volume 200 mL Add 150 mL microwave for 45 seconds Agitate the gel for 10 minutes Sensi...

Страница 51: ...mL Acetic Acid 60 mL Final Volume 600 mL see note above Fix the gel in 3 changes 200 mL each of fixing solution for 90 minutes Change the fixing solution every 30 minutes Sensitize Ultrapure water 105...

Страница 52: ...shaker UV transilluminator equipped with a standard camera or laser based scanner Staining Procedure 1 After electrophoresis remove the gel from the cassette page 24 and blot proteins onto nitrocellul...

Страница 53: ...able at www lifetechnologies com or contact Technical Support page 72 1 At the end of all staining and destaining steps wash the E PAGE Gel three times for 2 minutes each time in deionized water 100 m...

Страница 54: ...Gently smooth out any wrinkles in the assembly with a gloved hand or pipette 11 Place the plastic frame beveled side up on top of the cellophane Push the plastic clamps onto three edges of the frame T...

Страница 55: ...logies see the apparent molecular weights of these standards on E PAGE Gels listed under Assigned Apparent Molecular Weights to determine the apparent molecular weight of your protein You may need to...

Страница 56: ...2 3 4 5 6 7 8 9 E PAGE SeeBlue Pre stained Protein Standard Molecular Weight on E PAGE 96 6 Gel E PAGE MagicMark Western Protein Standard Molecular Weight on E PAGE 96 6 Gel 1 2 3 4 5 1 261 kDa 2 173...

Страница 57: ...sample and marker wells The gel was electrophoresed for 23 minutes using standard conditions Proteins were transferred to a 0 45 m nitrocellulose membrane using the semi dry blotting protocol describ...

Страница 58: ...8 Gel as described in this manual The gel contains the following samples lanes not indicated are blank Lane Sample M BenchMark Fluorescent Marker 5 L 2 8 13 19 26 32 37 43 Human kinase Lumio fusion pr...

Страница 59: ...an E PAGE 48 8 Gel and stained with SYPRO Ruby Protein Gel Stain as described in this manual The gel contains the following samples lanes not indicated are blank Lane Amount of BSA 1 2 2 5 ng 3 4 5 0...

Страница 60: ...contains the following samples lanes not indicated are blank Lane Sample M SeeBlue Plus2 Pre stained Standard 5 L 2 4 21 23 26 28 45 47 MagicMark XP Western Protein Standard 10 L 6 19 30 45 BenchMark...

Страница 61: ...lowing samples lanes not indicated are blank Lane Sample M SeeBlue Plus2 Pre stained Standard 5 L 2 4 21 23 26 28 45 47 MagicMark XP Western Protein Standard 10 L 6 19 30 45 BenchMark His tagged Prote...

Страница 62: ...er Staining Kit with are shown below BSA 0 5 1000 ng was run on an E PAGE 48 8 Gel and stained with the SilverQuest Silver Staining Kit as described in this manual The gel contains the following sampl...

Страница 63: ...The gel contains the following samples lanes not indicated are blank Lane Sample M SeeBlue Plus2 Pre stained Standard 5 L 2 4 21 23 26 28 45 47 MagicMark XP Western Protein Standard 10 L 6 19 30 45 B...

Страница 64: ...original plate Capture an image of the gel as described below and then use the E Editor 2 02 software to Align and arrange the lanes in the image Save the reconfigured image for further analysis Copy...

Страница 65: ...t correctly inserted into base Remove cassette and reinsert When the cassette is correctly inserted and power is on a fan in the base begins to run and a steady red light illuminates on the base page...

Страница 66: ...is running Estimate the amount of time remaining and then manually stop the run Protein bands distorted on membrane after semi dry blotting Non uniform electric field created around wells Be sure that...

Страница 67: ...ners See page 71 for ordering information Phosphoprotein Staining Protocol For all steps described below be sure to use sufficient volume of reagent to completely cover the gel using a suitable contai...

Страница 68: ...ine that falls within the excitation maxima of the stain 580 nm 7 Image the gel with a suitable camera with the appropriate filters using a 3 4 second exposure Results Results obtained using an E PAGE...

Страница 69: ...of reagent to completely cover the gel using a suitable container such that the gel moves freely 1 After electrophoresis remove the gel from the cassette page 24 and place the gel in a clean microwav...

Страница 70: ...m 8 Image the gel with a suitable camera with the appropriate filters using a 1 4 second exposure Results Results obtained using an E PAGE 48 8 Gel stained with Pro Q Sapphire 532 Oligohistidine Gel S...

Страница 71: ...cm one well to the next Space between Wells 4 5 mm Note E PAGE 48 8 Gels have a unique separation profile which gives protein resolution similar to that of a 4 12 Tris Glycine gel E PAGE 96 Gel Speci...

Страница 72: ...Temperature Ambient 15 C to 40 C Built in Features Digital time display 00 99 minutes alarm light LED The SBS Society for Biomolecular Screening standard 96 well plate format of the E Base fits on mos...

Страница 73: ...A The Mother E Base has been tested with up to 3 Daughter E Bases connected at one time Main plug is a disconnect device and must be easily accessible Do not attempt to open E Base devices To honor th...

Страница 74: ...re E PAGE Gels E Holder The E Holder Platform is used to hold an E PAGE Gel in place while loading Ordering information can be found on the following page E Editor 2 02 Software The E Editor 2 02 soft...

Страница 75: ...Transfer Buffer 20X 125 mL NP0006 NuPAGE Antioxidant 15 mL NP0005 NuPAGE Sample Reducing Agent 10X 10 mL NP0009 Nitrocellulose Filter Paper Sandwich 0 45 m 16 pk LC2006 Nitrocellulose Filter Paper Sa...

Страница 76: ...each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box...

Страница 77: ...73...

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