ER Stress-induced Autophagy ELISA Kit Pack

ER Stress-induced Autophagy ELISA Sampler Pack

ER Stress-induced Autophagy ELISA Sampler Pack from Assay Genie allows researchers to begin their investigation into ER Stress-induced Autophagy via analysing JNK, EIF2S1, anti-HSPA5, Beclin-1, ATG12 / APG12 in a 48 assay format, thus allowing researchers to identify and validate analytes within the pathway.

Pack Contents	Size	Reactivity	Sensitivity	Range	ELISA Type
Human JNK ELISA Kit	48 Assays	Human	0.188ng/ml	0.313-20ng/ml	Sandwich ELISA
Human EIF2S1 (Eukaryotic translation initiation factor 2 subunit 1) ELISA Kit (HUFI04476)	48 Assays	Human	0.094ng/ml	0.156-10ng/ml	Sandwich ELISA
Human anti-HSPA5 (anti-Heat shock protein family A member 5) ELISA Kit (HUFI08015)	48 Assays	Human	0.234ng/ml	0.391-25ng/ml	Indirect ELISA - Biotin
Human Beclin-1 ELISA Kit	48 Assays	Human	0.094ng/ml	0.156-10ng/ml	Sandwich ELISA
Human ATG12 / APG12 ELISA Kit	48 Assays	Human	0.094ng/ml	0.156-10ng/ml	Sandwich ELISA

Each individual ELISA kit in this pack will come with the following components:

Component	Quantity	Storage
ELISA Microplate (Dismountable)	4-12 strips	4°C for 6 months
Lyophilized Standard	1	4°C/-20°C
Sample/Standard Dilution Buffer	10ml	4°C
Biotin-labeled Antibody(Concentrated)	60ul	4°C (Protect from light)
Antibody Dilution Buffer	5ml	4°C
HRP-Streptavidin Conjugate(SABC)	60ul	4°C (Protect from light)
SABC Dilution Buffer	5ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	5ml	4°C
Wash Buffer(25X)	15ml	4°C
Plate Sealer	3	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 - g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to the wells, equilibrate the TMB substrate for at least 30 mins at 37°C. When diluting samples and reagents, ensure they are mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample (diluted at least Â« with Sample Dilution Buffer) and control (blank) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!
2.	Aliquot 100 µL of standard solutions into the standard wells.
3.	Add 100 µL of properly diluted Sample into the sample wells.
4.	Seal the plate with a cover and incubate at 37 °C for 90 mins.
5.	Wash: Repeat the aspiration/wash process for 2 times.
6.	Add 100 µL of Biotin-labelled antibody working solution into the bottom of each well (standard, test sample & zero wells) without touching the side walls.
7.	Seal the plate with a cover and incubate at 37 °C for 60 mins.
8.	Wash: Remove the cover, and wash plate 3 times with Wash buffer.
9.	HRP-Streptavidin Conjugate (SABC): Add 100?l of SABC Working Solution into each well, cover the plate and incubate at 37 °C for 30 minutes.
10.	Wash: Remove the cover and wash plate 5 times with Wash Buffer.
11.	TMB Substrate: Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark for 10-20 mins. (Note: This incubation time is for reference only, the optimal time should be determined by the end-user.) As soon as a blue colour develops in the first 3-4 wells (with most concentrated standards) and the other wells show no obvious colour, terminate the reaction.
12.	Add 50 µL of Stop solution into each well and mix thoroughly. The colour changes into yellow immediately.
13.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.