ab Mammalian Cell Lysis Buffer 5X Instructions for Use For mammalian cell lysis.

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1 ab Mammalian Cell Lysis Buffer 5X Instructions for Use For mammalian cell lysis. This product is for research use only and is not intended for diagnostic use. Version: 1 Last Updated: 30 October 2013

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3 Table of Contents Table of Contents 2 1. Overview 3 2. Protocol Summary 3 3. Kit Components 4 4. Storage and Stability 4 5. Materials Required, Not Supplied 4 6. Assay Protocol 5 7. Troubleshooting 6 2

4 1. Overview Abcam s Mammalian Cell Lysis Buffer 5X (ab179835) just requires a simple 5-fold dilution. 2. Protocol Summary Treat cells as desired, wash plate with PBS to remove residual media Lyse non-adherent cells with 1X Mammalian Cell lysis buffer to cell pellet Incubate at room temperature for min The cell lysate now is ready for further biochemical analysis 3

5 3. Kit Components Item Quantity Storage upon arrival Storage after use/ reconstitution Mammalian Cell Lysis Buffer 5X 10 ml 4 C 4 C 4. Storage and Stability Upon arrival, store at 4 C. Reagent is stable for at least 6 months if stored properly. 5. Materials Required, Not Supplied Petri dishes or well plates to grow cells Distilled water (ddh 2 O)or MilliQ PBS 4

6 6. Assay Protocol a) Warm up Mammalian Cell Lysis Buffer 5X to room temperature before use. b) Prepare 1X Mammalian Cell lysis buffer by adding 1 ml of Mammalian Cell Lysis Buffer 5X into 4 ml of MilliQ water, and mix them well. c) The 1X Mammalian Cell lysis buffer (from Step b) now is ready to use. d) Treat cells as desired, wash plate with PBS to remove residual media. e) The following table is the suggested volume for lysis of adherent cells. Plate 384-well 96-well 48-well 24-well 12-well 6-well 100cm 2 Volume 20 µl 50 µl 100 µl 150 µl 200 µl 300 µl 800 µl f) Lyse non-adherent cells by adding about 100 µl of 1X Mammalian Cell lysis buffer to 10 6 cell pellet. g) Incubate at room temperature for min. h) The cell lysate now is ready for further biochemical 5

7 7. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 6

8 Problem Reason Solution Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 7

9 Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit 8

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12 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 11 Copyright 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.

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