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    products/assay-kits/caspase-3-assay-kit-colorimetric-ab39401.pdf

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Cell Biology Apoptosis Intracellular Caspases etc Caspases
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Caspase-3 Assay Kit (Colorimetric) (ab39401)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (1)Q&A (11)References (275)

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Caspase-3 Assay Kit (Colorimetric) (ab39401)
  • Caspase-3 Assay Kit (Colorimetric) (ab39401)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 2 hr
  • Sample type: Cell Lysate, Tissue Lysate

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Overview

  • Product name

    Caspase-3 Assay Kit (Colorimetric)
    See all Caspase-3 kits
  • Detection method

    Colorimetric
  • Sample type

    Cell Lysate, Tissue Lysate
  • Assay type

    Enzyme activity
  • Assay time

    2h 00m
  • Product overview

    Caspase-3 Assay Kit (Colorimetric) ab39401 provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD.


    The Caspase-3 assay protocol is based on the formation of the chromophore p-nitroaniline (p-NA) by cleavage from the labeled substrate DEVD-pNA. The p-NA can be quantified using a spectrophotometer or a microtiter plate reader reading absorbance at 400 or 405 nm.


    Comparison of the absorbance of p-NA from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase-3 activity.


    Caspase-3 assay protocol summary:
    - add samples to wells
    - add reaction buffer and DEVD-p-NA substrate and incubate for 60-120 min at 37ºC
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K106 Caspase-3 Colorimetric Assay Kit. K106-100 is the same size as the 100 test size of ab39401.

    Due to the nature of the substrate, this assay also detects caspase-7 activity.

    Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells.

    Other caspase and apoptosis assays

    Review the full set of caspase assays, or the apoptosis assay and apoptosis marker guide.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    2X Reaction Buffer 4 x 2ml
    Cell Lysis Buffer 1 x 100ml
    DEVD-pNA 1 x 500µl
    Dilution Buffer 1 x 100ml
    DTT 1 x 400µl
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Caspases etc
    • Caspases
    • Cancer
    • Invasion/microenvironment
    • Apoptosis
    • Caspases
    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteolytic enzymes
    • Other proteases
    • Kits/ Lysates/ Other
    • Kits
    • Apoptosis Kits
    • Caspase Assays Kits
    • Caspase 3 assay kits
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Apoptosis
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • Caspases
    • Cancer
    • Cell Death
    • Apoptosis
    • Metabolism
    • Neuroscience
    • Processes
  • Function

    Involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a '216-Asp-
    -Gly-217' bond. Cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Involved in the cleavage of huntingtin.
  • Tissue specificity

    Highly expressed in lung, spleen, heart, liver and kidney. Moderate levels in brain and skeletal muscle, and low in testis. Also found in many cell lines, highest expression in cells of the immune system.
  • Sequence similarities

    Belongs to the peptidase C14A family.
  • Post-translational
    modifications

    Cleavage by granzyme B, caspase-6, caspase-8 and caspase-10 generates the two active subunits. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. Active heterodimers between the small subunit of caspase-7 protease and the large subunit of caspase-3 also occur and vice versa.
    S-nitrosylated on its catalytic site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway, associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active site thiol.
  • Cellular localization

    Cytoplasm.
  • Target information above from: UniProt accession P42574 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • A830040C14Rik
    • Apopain
    • CASP 3
    • CASP-3
    • CASP3
    • CASP3_HUMAN
    • Casp3a
    • Caspase 3
    • Caspase 3, apoptosis-related cysteine peptidase
    • Caspase 3, apoptosis-related cysteine protease
    • Caspase 3, apoptosis-related cysteine protease a
    • Caspase-3 subunit p12
    • Caspase3
    • CC3
    • CPP 32
    • CPP-32
    • CPP32
    • CPP32B
    • Cysteine protease CPP32
    • EC 3.4.22.56
    • ICE3
    • LICE
    • mldy
    • OTTHUMP00000165052
    • OTTHUMP00000165053
    • OTTHUMP00000165054
    • PARP cleavage protease
    • Procaspase3
    • Protein Yama
    • SCA 1
    • SCA-1
    • SCA1
    • SREBP cleavage activity 1
    • Yama
    • Yama protein
    see all

Associated products

  • Corresponding Antibody

    • Anti-Caspase-3 antibody (ab13847)
    • Anti-Cleaved Caspase-3 antibody (ab2302)
    • Anti-Cleaved Caspase-3 antibody [E83-77] (ab32042)
    • Anti-Caspase-3 antibody (ab4051)
  • Related Products

    • Z-D(OMe)E(OMe)VD(OMe)-FMK, Cell permeable caspase-3 inhibitor (ab120488)
    • Z-VDVAD-FMK, caspase-2 inhibitor (ab142035)
    • Q-DEVD-OPh, caspase-3 inhibitor (ab142037)
    • PAC-1, caspase-3 activator (ab142074)

Images

  • Caspase-3 Assay Kit (Colorimetric) (ab39401)
    Caspase-3 Assay Kit (Colorimetric) (ab39401)Image from Xu G. et al. J Biol Chem. 2012;287(7):4808–4817. doi:10.1074/jbc.M111.275719. Fig. 2G.
    H9c2 cells were either untreated (control) or treated with 2µg/ml of recombinant lipocalin-2 (Lcn2) for 24h. Cell lysates were assayed for caspase-3 activity (n = 3, *, p<0.05). Image obtained from Xu G et al., JBC, 2012 Feb 11;28(7):4808-17
  • Caspase-3 Assay Kit (Colorimetric) (ab39401)
    Caspase-3 Assay Kit (Colorimetric) (ab39401)

    Caspase-3 in Jurkat lysates (3.3 x10e6 cells) following 20 hour exposure to 2 uM Camptothecin (ab120115) or 10 ng/mL anti-Fas Ab (MBL).

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (275)

Publishing research using ab39401? Please let us know so that we can cite the reference in this datasheet.

ab39401 has been referenced in 275 publications.

  • Aring L  et al. A neurodegeneration gene, WDR45, links impaired ferritinophagy to iron accumulation. J Neurochem 160:356-375 (2022). PubMed: 34837396
  • Lan X & Xu Y Protective role of lidocaine against cerebral ischemia-reperfusion injury: An in vitro study. Exp Ther Med 23:42 (2022). PubMed: 34849157
  • Dung DTM  et al. Design, synthesis and evaluation of novel 2-oxoindoline-based acetohydrazides as antitumor agents. Sci Rep 12:2886 (2022). PubMed: 35190616
  • Lai MC  et al. The Citrus Flavonoid Hesperetin Encounters Diabetes-Mediated Alzheimer-Type Neuropathologic Changes through Relieving Advanced Glycation End-Products Inducing Endoplasmic Reticulum Stress. Nutrients 14:N/A (2022). PubMed: 35215394
  • Liu W  et al. OTUD1 stabilizes PTEN to inhibit the PI3K/AKT and TNF-alpha/NF-kappaB signaling pathways and sensitize ccRCC to TKIs. Int J Biol Sci 18:1401-1414 (2022). PubMed: 35280681
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 12 Abreviews or Q&A

It doesn't work for tissue samples

Inconclusive Good 4/5 (Ease of Use)
Abreviews
Abreviews
abreview image
We tried to quantify Caspase 3 activity in mouse mammary gland during mammary involution after lactation. We tested different protein amounts fron 50 to 200 microgrames (as indicated in protocol booklet), but the product did not work in any case. We wanted to compare Caspase 3 activity in three samples at different timepoints of involution, and we were not able to observe differences (A), which really exist, as we confirmed by western blot of the same samples (B). We checked the kit was not faulty testig in HC11 mammary cell line, inducing apoptosis with cisplatin 15 ug/ml for 24h (C).
The technical service told us it was a problem with our samples and the did not offer a way to solve our problem.
Then, we found a fluorimetric assay ab39383, which is exactly the same but with a fluorometric substrate instead of a colorimetric one, which is more sensitive as abcam scientific support says in the questions and answer section of the product wall in their website. We aquired the fluorometric substrate and repeated the assay with the same samples. This time, the product worked perfectly (D).
So, for mouse mammary gland tissue samples, I would not recommend this product. It seems it has not enough sensitivity. I would try the fluorimetric assay.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Mr. Adrian Blanco

Verified customer

Submitted Jul 17 2014

Question

The datasheet for this product says that it can be used for tissue extracts and cell lysates. We have fresh frozen ground squirrel (Ictidomys tridecemlineatus) brain tissue (flash frozen in liquid nitrogen), currently stored at -80 degrees.

Is the fact that the samples were frozen a potential problem? The protocol seems to indicate that you should start with fresh tissue.

Read More

Abcam community

Verified customer

Asked on May 02 2013

Answer

Freezing and thawing may partially degrade caspase activity but the protocol, in step 1.2., suggests it will not be a problem: "Transfer supernatant (cytosolic extract) to a fresh tube and put on ice for immediate assay or aliquot and store at -80°C for future use."

Read More

Abcam Scientific Support

Answered on May 02 2013

Question

Inquiry: I'd like to know if the Caspase 3 Assay Kit (Colorimetric) (ab39401) is suitable for bovine cells. Price? Thanks a lot in advance!

Read More

Abcam community

Verified customer

Asked on Apr 27 2013

Answer


ab39401 should recognize all mammalian forms of Caspase 3. One kit is $470, which provides enough solution for one 96 well plate.

Read More

Abcam Scientific Support

Answered on Apr 27 2013

Question

I want to assay caspase 3 activity in platelet rich plasma (PRP) during storage. Do i use abcam caspase 3 colorimetric assay kit for this purpose? Lysis buffer in the kit is suitable for platelet lysis ? If you have any protocol to assay platelet caspase 3 activity by this kit, please send me, beacuse i want to use this kit for my project.  

Read More

Abcam community

Verified customer

Asked on Apr 22 2013

Answer

This kit is good just for cell and tissue lysates but not plasma. If the you are able to collect the platelets from the plasma in a clean manner you will be able to use the kit with the recommended protocol.

Read More

Abcam Scientific Support

Answered on Apr 22 2013

Question

The customer asking if this kit can work on serum or not?
Caspase 3 Assay Kit (Colorimetric) (ab39401)
I checked all caspase 3, and all of them in Abcam bot written to work on Serum?
Truly yours,

Read More

Abcam community

Verified customer

Asked on Jan 21 2013

Answer

Thank you for contacting us.

This kit has not been tested with serum sample yet, so we can provide Abtrial discount if customer is interested in testing this kit.

Please note that this product is "for research use only and are not intended for diagnostic or therapeutic purpose.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Abcam Scientific Support

Answered on Jan 21 2013

Question

Dear Technical Team,

Hope this e-mail finds you well!

Since, it is not mentioned in the protocol of Caspase 3 Assay Kit (Colorimetric) (ab39401). Kindly suggest us on how to proceed with tissues or how to make single cell suspension as desired by the customer.

Looking forward towards your valued response on it.

With Best Regards,

Read More

Abcam community

Verified customer

Asked on Dec 05 2012

Answer

Thank you for contacting us and your interest in our products.

The Caspase 3 Assay Kit (Colorimetric) (ab39401) can be used with tissue samples and we would suggest following the following protocol:

1. Induce apoptosis in tissue (10-100) mg by desired method. Concurrently have control without induction.

2. Re-suspend tissue in 50-400 μl of chilled Cell Lysis Buffer and homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope.

3. Centrifuge for 1 min in a microcentrifuge (10,000 x g).

Subsequently follow the remaining protocol as described with cell samples.
I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

Read More

Abcam Scientific Support

Answered on Dec 05 2012

Question

Dear.

I am writing to you because I am interested in the ab39401.
We investigated the caspase-3 activity inin vivo.
So, I wonder if the celllysisbuffer of ab39401couldapply tomouse tissue for our study.

If you have any questions, please contact me. I will reply you as soon as possible.
I am looking forward to your earliest reply.

Yours sincerely

Read More

Abcam community

Verified customer

Asked on Sep 19 2012

Answer

Thank you for your enquiry.

I am pleased to confirm that this kit (ab39401 Caspase 3 Assay Kit (Colorimetric))is compatible with mouse samples. Here are the references we have from this kit. I hope this will provide some reassurance that this kit is good for a huge array of mammalian samples, including the mouse samples.

I hope this will be helpful. Please do not hesitate to contact me if you have any further questions.


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- Caporali, S., et al. (2003) Mol. Biol. Cell 14:5051-5059.

- Carley Glass et. al. MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart. Am J Physiol Heart Circ Physiol, Nov 2011; 301: H2038 - H2049.

- Cartee, L., et al. (2002) Mol. Pharmacol. 61:1313-1321.

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- Chen T-H et al (2008) Clin. Cancer Res. 14: 4250- 4258.

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- Daniel, D., et al. (2004) Blood 10.1182/blood-2003-02-0635.

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- Ding, H.F., et al. (2000) J. Biol. Chem. 275:38905-38911.

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- Farhana L et al (2009) Mol. Cancer Ther.; 8: 1625 - 1635.

- Feinman, R. et al. HIF-1 Mediates Pathogenic Inflammatory Responses to Intestinal Ischemia-reperfusion Inhury. Am. J. Physiol. Gastrointest. Liver Physiol., 2010; 299: G833-G843

- Fujii T., et al. (2000) J. Biol. Chem. 275:7574-7582.

- Fukutomi, Y. et. al. Apoptosis-Inducing Activity of Clofazimine in Macrophages. Antimicrob. Agents Chemother., Sep 2011; 55: 4000 - 4005.

- Galindo, C.L., et al. (2004) J. Biol. Chem. 279:37597-37612.

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- Han W et al. (2007) Mol. Cancer Ther. 6: 1641-1649.

- Hojman, P. et. al. Exercise-induced muscle-derived cytokines inhibit mammary cancer cell growth. Am J Physiol Endocrinol Metab, Sep 2011; 301: E504 - E510.

- Hu Y. et al. (2006) J. Immunol. 177: 8522-8530.

- Idogawa M et al (2009) Clin. Cancer Res.; 15: 3725 - 3732.

- Idogawa M et al (2009) Clin. Cancer Res; 15: 3725 - 3732.

and 15-Lipoxygenases. Cancer Prevention Research, Sep 2010; 3: 1132 - 1140.

- Kawakami, M., et al. (2002) Mol. Cancer Thep. 1:999-1007.

- Kenta Moriwaki et al., GDP-mannose-4,6-dehydratase (GMDS) Deficiency Renders Colon Cancer Cells Resistant to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor- and CD95-mediated Apoptosis by Inhibiting Complex II Formation, J. Biol. Chem., Dec 2011; 286: 43123 - 43133.

- Khabbaz KR et al. (2008) J. Thorac. Cardiovasc. Surg. 135: 139 - 146.

- Kim L. and Denkers E.y. (2006) J. cell Sci. 119: 2119-2126.

- Kim SY et al (2008) Blood 111: 5704-5711

- Kim, J. W. et. al. The Role of the 3D Environment in Hypoxia-induced Drug and Apoptosis Resistance. Anticancer Res, Oct 2011; 31: 3237 - 3245.

- Konishi, T., et al. (2005) Mol. Cancer Ther. 4: 743-750.

- Kown, M.H., et al. (2000) Circulation. 102:228-232.

- Lamontagne, L. et al. Hepatits B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal, Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis. J. Virol. 2010; 84: 907-917

- Lee, S-Y, et al. (2006) Infect. Immun. 10.1128/IAI.01700-06.

- Leu, S. W. et. al. TLR4 through IFN-β Promotes Low Molecular Mass Hyaluronan-Induced Neutrophil Apoptosis. J. Immunol., Jan 2011; 186: 556 - 562.

- Li Y., et al. (2007) Diabetes 56: 656-665.

- Li, C., et al. (2005) J. Biol. Chem. 280: 26193-26199.

- Li, L., et al. (2005) Toxicol. Sci. 86: 116-124.

- Liao, Y. et al. miR-214 Regulates Lactoferrin Expression and Pro-Apoptotic Function in Mammary Epithelial Cells. J. Nutr., Sep 2010; 140: 1552 - 1556.

- Lieman, J.H., et al. (2005) J. Biol. Chem. 280: 10484-10490.

- Macchi, B. et al. The Novel Proapoptotic Actvity of Nonnatural Enantiomer of Lentiginosine. Glycobiology 2010 20: 500-506.

- Machiya J-I et al. (2007) Am. J. Respir. Cell Mol. Biol. 36: 418-426.

- Maity, B. et. al. Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity. J. Biol. Chem., Jan 2011; 286: 1409 - 1419.

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- Oh, P. S. et. al. Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells. Am J Physiol Gastrointest Liver Physiol, Aug 2011; 301: G347 - G355.

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- Toyonaga, J. et. al. Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production. Nephrol. Dial. Transplant., Jan 2011; 10.1093/ndt/gfq750.

- Toyonaga, J. et. al. Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production. Nephrol. Dial. Transplant., Aug 2011; 26: 2475 - 2484.

- Toyonaga, J. et. al. Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production. Nephrol. Dial. Transplant., Aug 2011; 26: 2475 - 2484.

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Abcam Scientific Support

Answered on Sep 19 2012

Question

Hello, Abcam Tech Support,   I would like to know bout the following issues for my purchase of this kit, Caspase 3 assay kit, ab39401.   1 Does this kit recognize rat caspase-3? 2 Does this kit recognize both activated and non-activated caspase-3 or either? 3 Can I use this kit for tissue caspase 3 assay? If so, a 50-200 mcg protein use remains same as a recommended protocol? 4 For tissue assay, any additional suggestions on the protocol?   Thank you in advance for your reply,  

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Abcam community

Verified customer

Asked on Sep 04 2012

Answer

Thank you for contacting us. I have answered each of your questions below. Please let me know if you have any additional questions about this or any Abcam product.

1. Does this kit recognize rat caspase-3?

This will recognize all mammalian forms (90% homology of Caspase 3 w/rat)



2. Does this kit recognize both activated and non-activated caspase-3 or either?

The assay is based on spectrophotometric detection of thechromophore p-nitroaniline (p-NA) after cleavage from the labeledsubstrate DEVD-p-NA. Therefore it should only recognize activated Caspase 3.



3. Can I use this kit for tissue Caspase 3 assay? If so, a 50-200 mcg protein use remains same as a recommended protocol?

Yes, you can use tissue lysates for this kit. Use exactly the same protocol. Use 200-400 µl of the lysis buffer for ach 10 mg of tissue. Samples should be completely homogenized, preferably using a Dounce homogenizer. Some substances can interfere with the assays and should be avoided in sample preparation. Please see individual kit datasheets for more specific information on which substances may affect the specific kits, but in general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) are known to interfere with these assays and should be avoided in sample preparation. Moreover, we recommend deproteinating the sample prior analysis using a 10kD spin column (ab93349).



4. for tissue assay, any additional suggestions on the protocol?

Use exactly the same protocol. Use 200-400 µl of the lysis buffer for ach 10 mg of tissue.






I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam Scientific Support

Answered on Sep 04 2012

Question

What step is the Dilution buffer used for? Is it for diluting the samples for the protein quantification step?

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Abcam community

Verified customer

Asked on Jul 10 2012

Answer

Thank you for contacting Abcam.

The dilution buffer is to dilute the final samples before reading their absorbance, in case of the undiluted readings being above the detection range of the instrument.
Hope this information has been helpful for you.
Please let me know if you have any other questions.

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Abcam Scientific Support

Answered on Jul 10 2012

Question

I order your ab39401 caspase 3 assay kit.
In the kit the is a dilution buffer, but in the protocol the dilution
buffer is not mentioned.
I suppose it is to dilute lysate protein in step 7 (page 6 in
instruction for use)? Or there is any function for this buffer? Thank
you.

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Abcam community

Verified customer

Asked on May 14 2012

Answer


The dilution buffer is used in case you do not have a plate reader an need to read the concentration manually in a cuvette: Step10. "Read samples at 400 or 405 nm in a microtiter plate reader, or
spectrophotometer using a 100 μl micro-quartz cuvette, or dilute
sample to 1 ml with Dilution Buffer and using regular cuvette."

In step 7, you are correct, the protein amount in the samples is measured and than adjusted with the lysis buffer for the assay.

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Abcam Scientific Support

Answered on May 14 2012

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